Flow Cytometry Innovative Solutions

Innovative Solutions for Flow Cytometry Analysis Optimized Kits and Reagents

EMD Millipore is a division of Merck KGaA, Darmstadt, Germany

Guava integrated flow cytometry solutions ®

Flow cytometry is an essential tool for in-depth cell

flow cytometry solution includes our instruments,

analysis. With the capacity to simultaneously

assays, and software—as well as the cell

measure multiple parameters on hundreds of

isolation and culture tools to prepare your samples.

individual cells per second, flow cytometry offers

With a flow cytometry solution right in your lab,

greater speed, precision, and detail than most other

you’ll experience superior performance, higher

cell analysis methods available to scientists today.

quality data and faster progress from hypothesis to

Integrated products from EMD Millipore will help

results.

streamline your workflow. Our complete benchtop

What’s inside...

Cell Health

Cell Signaling

Stem Cells

• Cell Counting & Viability

• MAPK Pathway

• Embryonic Stem Cell

• Cell Cycle

• EGFR Pathway

• DNA Damage

• PI3/Akt/mTOR Pathway

• Mitochondrial Analysis

• Jak/STAT Pathway

• Apoptosis

• Chemokine

Immunology

Milli-Mark® Conjugated Antibodies

• Regulation T-Cell • Phenotype Markers

(Human/Mouse) • Neural Stem Cell (Rodent)

Components of the guava® Flow Cytometry Solution • Instruments: easyCyte™ Flow Cytometers • Software • Kits & Reagents

2

Kits and Reagents Advantages • Multiplexing capabilities • Easy to use, with fewer incubation and wash steps • Fully validated and concentration-optimized, guaranteed to work in flow cytometry

FlowCellect™ Kits and Milli-Mark Conjugated Antibodies

FlowCellect Kits

Milli-Mark Conjugated Antibodies

Many researchers invest time

FlowCellect kits are EMD Millipore’s

Milli-Mark antibodies are directly

optimizing and validating antibodies

proprietary multiparameter flow

conjugated primary antibodies

for flow cytometry, only to discover

cytometry kits for the analysis

that are validated for flow

that these antibodies do not perform

of cellular events and/or cell

cytometry in addition to traditional

well when multiplexed together,

phenotypes. Each kit has unique

applications like Western blotting

because of interference from the

combination of directly conjugated

and immunocytochemistry (see

matrix or from other antibodies.

antibodies, and/or fluorescent dyes

Figure 1B for an example of antibody

and protein reporters to monitor

validation). Because of their

EMD Millipore’s FlowCellect kits

changes in protein expression and

extensive crossplatform validation,

and Milli-Mark conjugated primary

posttranslational modification. The

Milli-Mark antibodies are valuable,

antibodies are fully optimized

kits also contain complete buffer

convenient building blocks with

for fast, easy, and accurate

sets, protocols and pre-defined gate

which you can configure your own

multiparametric flow cytometry.

settings. They are fully optimized for

assays.

We’ve taken the guesswork out of

“plug-and-play” cellular analysis on

assay development so you can focus

guava instruments and other flow

on your results. We optimize and

cytometers. Using a four-step validation process

are cells and a research question; our

to develop our FlowCellect kits, we’ve

assay kits will do the rest, and you’ll

eliminated the need to design your

have data before your cells are ready

experiment or optimize antibodies

to split again.

and buffers (Figure 2 below).

B. 250

250

130 100 70 55

130 100 70 55

70kDa 44kDa

35 27 15 10

35 27 15 10 OCT-4 antibody

SSEA-4 antibody

Figure1. (A) Mouse embryonic stem cells and fibroblasts labeled with Oct-4 (green), SSEA-1 (red), and Hoechst nuclear stain (blue). (B) Human embryonic stem cell lysate.

validate every antibody, making sure they work together well. All you need

A.

Figure 2. FlowCellect Kit Four-step Validation Process.

Antibody Conjugation and Testing

Multiplexing, Stability, Performance Claims

Antibodies are screened

Antibodies are conjugated

The antibody conjugates

each kit are carefully

primarily by Western blot

to compatible

are optimized to provide

selected by reviewing

to determine specificity,

fluorophores that will

the best signal-to-noise

many publications.

then validated for flow

ensure less fluorescence

ratio when multiplexing.

cytometry using a

spectrum overlap.

Antibody Selection and Assay Design

Antibody Component Validation

The antibodies used in

secondary antibody.

3

Cell Health Discover the power of flow cytometry for multiparametric cell health analysis Cell Counting and Viability

Knowing the performance profile of your cells prior to running your bioassay can mean the difference between

Cell counting and viability assessments can be used in a

valid assay results and wasted reagents, lost time and

variety of applications, such as cytotoxicity studies, PBMC

discarded data.

counting and rapid apoptosis assessment.

Monitoring key indicators of cell health and performance,

guava ViaCount® Assay Kits

such as the apoptotic fraction and stage of apoptosis,

The guava ViaCount assay is fast becoming the new

viability, cell cycle, cell counts, transfection efficiency, and

standard for viability and cell counting. In this simple

target expression levels, helps establish uniform standards

no-wash, mix-and-read-assay, you can accurately obtain

of cellular performance across long-term research studies.

absolute total cell counts, perform viability assessments and determine apoptotic percentages—all from tiny

These standards can be applied to a wide range of

samples. You’ll enjoy several advantages over traditional

bioassays. Whether you are establishing screen/no screen

methods, including greater accuracy, reproducibility and

criteria for high throughput screening, monitoring and

speed.

optimizing bioreactor conditions, or eliminating sources of assay variability, consistent monitoring of your cell model

Advantages

improves your bioassay performance and productivity.

Assays: • Simple no-wash, mix-and-read procedure • Counts up to 10 times faster than manual methods

103

Dead

102 101

• More reproducible than traditional tests

104 Live Viability Stain

Nucleated Cells

104

Apoptotic

100 100

101 102 103 Viability Stain

104

Samples:

Dead

103

• Uses small samples in tubes or a 96-well plate

102 101

• Handles low-density and small-volume cell samples

Live

100 100

Apoptotic 101 102 103 Annexin V

• Works with adherent or suspended cells and mammalian and insect cells

104

T he blue population of cells show a significant amount of annexin V staining (right plot), indicating that intermediate levels of staining with the viability dye correlates with apoptosis.

guava ViaCount Assay Kits Description

4

Qty

Catalog No.

Guava ViaCount Reagent Kit • The new standard for cell counting and viability assessment.

100 tests

4000-0040

600 tests

4000-0041

Guava ViaCount Flex Reagent Kit • Non-mammalian cell lines (ie. SF9 insect cells). • Low density cell samples (~104 cells/mL). • Cell lines that stain heterogeneously.

100 tests

4500-0110

500 tests

4700-0060

Guava ViaCount Cell Dispersal Reagent Kit (CDR) • Uses enzymes to gently disaggregate clumped cells in suspension, improve the accuracy and precision of cell counts.

100 tests

4700-0050

Discover the power of flow cytometry for multiparametric cell cycle research. Cell Cycle

FlowCellect Cell Cycle Kits

The cell cycle can be divided into two distinct stages. The

EMD Millipore has developed and optimized two bivariate

first stage is interphase which consists of the G1, S, and

cell cycle analysis kits using phase-specific antibodies

G2 phases, in which cells are active, growing, and DNA is

in addition to a DNA dye. Bivariate analysis will not only

being replicated. The second is M phase, also known as the

reveal the cell distribution within a particular phase of

“mitotic phase”, in which cell division takes place.

cell cycle, but can also enable the researcher to elucidate mechanisms of cell cycle regulation, without sophisticated

Cell cycle phase distributions can be used to assess cell

software modules or algorithms.

health, proliferation, as well as the potential mechanism • Quantitative measurements of percentage of cells within each cell subpopulation

phase cells can help identify cells undergoing mitosis. Flow cytometry analysis of cell DNA content has been

distribution. However, the limitation of single-marker analysis, such as a DNA dye only, is that cells within each phase cannot accurately be determined without

Cell Growth

• Includes all optimized flow cytometry antibodies and buffers • No specific cell cycle analysis software required

INTE HAS

to be widely used for the estimation of cell cycle phase

• Minimal assay development needed

Cells that cease dividing

RP

one of the best and most popular tools for researchers

Mitosis

E

synthesized DNA. Also, distinguishing cells in G2 from M

esis

population of S phase cells can reflect the amount of newly

okin

Advantages

Cyt

of antineoplastic agents. For example, measuring the

Cell Prepares for Division DNA Synthesis

mathematical interpolation using analysis software.

featured product FlowCellect Bivariate Cell Cycle Kit for DNA Replication Analysis accuracy and confidence. The kit includes a directly conjugated Anti-BrdU Alexa Fluor® 488 antibody plus a DNA dye (propidium iodide). BrdU incorporation is a widely accepted method of measuring DNA replication and kinetics of cell cycle progression. The percentage of BrdU labeled cells is a reliable estimate of the S phase compartment, and labeled cells can then be followed through the cell cycle.

S Phase

103

BrdU

Investigate DNA replication in the S phase with high

104

102 101 100

G1 0

1

G2/M 3

5

7

Propidium Iodide (x1000)

9

Detection of DNA replication by analysis of S phase cells. Bivariate flow cytometric analysis using BrdU Alexa Fluor 488 conjugate can distinguish S phase cells with great accuracy, not only based on their difference in DNA content from G1 or G2/M cells but also as having incorporated BrdU. G=24% (-BrdU, 1X DNA content) S=72% (↑BrdU, 1-2X DNA content) G2/M=4% (-Brdu, 2X DNA content) 5

FlowCellect Bivariate Cell Cycle Kit for G2/M Analysis (Application) Control (untreated) 3% cells in M phase

103

102

101

100

This kit uses the nuclear DNA stain, propidum iodide (PI), to measure cell cycle. Resting cells (G0/G1) contain two copies

Nocodazole treated

pH3-Alexa Fluor 488

pH3-Alexa Fluor 488

pH3

104

guava Cell Cycle Assay

104 18% cells in M phase

of each chromosome. Cycling cells synthesize chromosomal

103

intensity. When all chromosomal DNA has doubled (G2/M

DNA (S phase), which results in increased fluorescence phase), cells fluoresce with twice the intensity of the initial population.

102

101

0

1

3

5 PE (x1000)

7

9

100

0

1

3

5 7 PE (x1000)

9

Propidium Iodide Nocodazole treatment increases percentage of cells in M phase. Cell were either treated with 100 µm Nocodazole (test sample) or left untreated (control) overnight at 37º C. By plotting the phosohorylation of histone H3 at Ser10 (y axis) versus DNA content (x axis), an increase in the proportion of G2/M cells was observed indicating that mitotic cells have accumulated after treatment. Apporomately 2% of cells reside in M phase under normal conditions in Jurkat cells, but when treated cell population increased 18%.

featured product 104

Investigate the G2/M phase transition with high accuracy

103

and confidence. The phosphorylation of histone H3 at Ser10 correlates with the G2 to M phase transition and is a prerequisite for chromatin condensation at mitosis. At the end of mitosis, histone H3 is rapidly dephosphorylated and remains unphosphorylated throughout the remainder

pH3-Alexa Fluor 488

FlowCellect Bivariate Cell Cycle Kit for G2/M Analysis

M

102 G2

G1

S

101

of interphase. Therefore, phospho-histone H3 (Ser10) is a reliable, specific marker of M-phase cells.

100

0

1

3

5 P1 (x1000)

7

9

Cell Cycle Phases: G1 = 57% S = 19% G2 = 15% M = 3%  iscrimination between G2 and M phase cells by measuring D the phosphorylation of histone H3 on Ser10. Histone H3 is constitutively phosphorylated at Ser10 during metaphase.

FlowCellect Cell Cycle Kits Description

6

Qty

Catalog No.

Bivariate Cell Cycle Kit for DNA Replication Analysis Anti-BrdU / Propidium Iodide Solution

25 tests

FCCH025102

Bivariate Cell Cycle Kit for G2/M Analysis Anti-phospho-Histone H3(Ser10) / Propidium Iodide Solution

25 tests

FCCH025103

guava Cell Cycle Reagent Propidium Iodide Solution

100 tests

4500-0220

DNA Damage Signaling Pathway

FlowCellect DNA Damage Histone H2A.X Dual Detection Kit

Investigating the DNA damage signaling pathway is an

FlowCellect Histone H2A.X DNA Damage Dual Detection kit

important area for genome health and cancer research.

includes two directly conjugated antibodies, a phospho-

Evidence suggests there is a direct correlation between

specific Anti-phospho-Histone H2A.X (Ser139)-PerCP

DNA damage and cell cycle. Cells that are defective in

and an Anti-Histone H2A.X-FITC conjugated antibody to

DNA damage pathways can cause cancer because they

measure total levels of Histone H2A.X. This two color kit is

lack the ability to sense and repair the damage, leading to

designed to detect the extent of Histone H2A.X pathway

genetic instability and ultimately uncontrolled cell growth.

activation by measuring H2A.X phosphorylation relative to

The main kinase activated in response to double-stranded

the total H2A.X expression in any given cell population. By

DNA breaks is ATM or Ataxia telangiectasia mutated

doing such, the levels of both the total and phosphorylated

kinase. ATM is a member of the phospho inositide 3-kinase

protein can be measured simultaneously in the same cell,

(PI3K)-related Ser/Thr protein kinase family. Inactive ATM

resulting in a normalized and accurate measurement of

exists as a dimer but quickly dissociates and becomes

H2A.X activation after stimulation. Moreover, simultaneous

phosphorylated on Serine 1981 in response to ionizing

measurement of both total and phospho-Histone H2A.X

radiation. Once activated, ATM phosphorylates a number

confirms target specificity of the phosphorylation event.

of downstream factors, including P53, CHK2, SMC1, NBS1,

Together, a total and phospho antibody duo performed

and Histone H2A.X.

in multiplex provides an enhanced and more reliable Changes in Chromatin Structure

Ionizing Radiation

featured product

detection of the phospho: total ratio within a mixed cell population. Using this antibody pair provides a sensitive and valuable tool to study the factors that induce DNA damage and/or affect DNA repair, and allow one to explore the linkage between DNA damage, cell cycle checkpoints,

MRN Complex

and initiation of apoptosis. ATM

P

BRCA1 ATM P

Data below: Unstimulated HeLa cells are stained with both phospho-Histone H2A.X-PerCP and Anti-Histone H2A.X-FITC (A), where there is no indication of Histone H2A.X activation via phosphorylation, but only on total H2A.X as noted by 97.2% of cells. However, once HeLa cells were stimulated with 100 µM etoposide, simultaneous measurement of both total and phospho Histone H2A.X confirms target specificity of the phosphorylation event as indicated by the double positive cell population (B) as indicated by the 2.09% to 97.15% increase of double positive staining. The levels of both the total and phosphorylated protein can be measured simultaneously in the same cell, resulting in a normalized and accurate measurement of H2A.X activation after stimulation.

A. Unstimulated

ATM

CHK2

B. Stimulated

P P

P

53BP1 P P

MDC1

P

SMC1 P

P

p53

P

P

P

Cell-cycle Checkpoint Arrest

DNA Repair

pH2AX-PerCP (RED-H_cg)

Phospho H2AX - PerCP

P

H2AX

pH2AX-PerCP (RED-H_cg)

P

H2AX-FTC (GRN-HLog)

H2AX-FTC (GRN-HLog)

Total H2AX - FITC Apoptosis

FlowCellect DNA Damage Kits Description

Multicolor DNA Damage Response Kit

Qty

Catalog No.

25 Tests

FCCH025104

25 tests

FCCS025153

25 tests

FCCH025142

25 tests

FCCH025143

Anti-p-SMC1 (S957)- Alexa Fluor 488/ Anti-pATM (S1981)- PE/ Anti-pHistoneH2A.X(S139)- PerCP

DNA Damage Histone H2A.X Dual Detection Kit Anti-pHistone H2A.X (Ser139)-PerCP/ Anti-Histone H2A.X-FITC

Cell Cycle Checkpoint H2A.X DNA Damage Kit Anti-pHistone H2A.X (Ser139), clone JBW301 - Alexa Fluor 488/ Propidium Iodide Solution

Cell Cycle Checkpoint ATM DNA Damage Kit Anti-pATM (Ser1981), clone 10H11.E12 - Alexa Fluor 488/ Propidium Iodiden

7

Discover the power of flow cytometry for multiparametric mitochondrial analysis. Mitochondrial Analysis

FlowCellect Mitochondrial Kits

Mitochondria are critical cellular organelles that produce

These kits harness the power of flow cytometry to assess

90% of cellular energy, control cell survival by regulating

changes in mitochondrial membrane potential, apoptosis

apoptosis, and produce reactive oxygen species (ROS).

as measured by Annexin V binding, mitochondrial oxidative

Mitochondrial superoxide generation results in oxidative

stress, and cell death, using only minimal cellular samples.

stress, damage and cell death by apoptosis or to cellular

The kits may be used with most dual laser flow cytometry

energetic decline. Therefore, mitochondrial dysfunction

systems equipped with a 488 nm and a 644 nm laser.

caused by disease or compound treatment has dire Advantages

consequences that can result in cell death.

Assay: Monitoring impact on mitochondria and related cell health

• Multiplex detection with no optimization required

markers is an important part of drug screening programs,

• Highly reproducible

pathway mapping, apoptosis, and disease research.

• Minimal assay development needed

Flow cytometry detects multiple markers simultaneously at

• All optimized flow cytometry antibodies and buffers included

various stages of apoptosis, making it a powerful technique

• Enables novice users to perform complex analysis

for studying pathways governing cell health and cell death.

Samples: • Designed to run 100 samples of human cells Extrinsic Pathway Signal

FAS

Pro-Caspase 8 Mitochondrial Signaling and Apoptosis

ER Stress, DNA Damage, Oxidants

Adapted from Bayir and Kagan Critical Care 2008 12:206.

Bax

Bak

t-Bid

Mitochondria

Caspase 8

Smac/Diablo AIF Endo G

∆ψm Mitochondrial Potential Change IAP

APAF-1

Pro-Caspase 9 Cyt c

Apoptosome

Cyt c

Activated Caspase Cascade

Chromatin Condensation

IAP

Caspase 3 Nucleus

DNA Fragmentation

8

Bid

Apoptosis

featured product FlowCellect MitoDamage Kit

Kit Component

These kits harness the power of flow cytometry to assess

MitoSense Red Dye

Red

changes in mitochondrial membrane potential, apoptosis

Annexin V-CF488A

Blue

7-AAD

Blue

as measured by Annexin V binding, mitochondrial oxidative

Laser

Buffer Pack 10X Assay Buffer HSC

stress, and cell death, using only minimal cellular samples. The kits may be used with most dual laser flow cytometry systems equipped with a 488 nm and a 644 nm laser. Simultaneously measures 3 important cell health parameters:

The FlowCellect MitoDamage kit can thus distinguish multiple populations:

• Change in mitochondrial potential (considered an early

1. Healthy cells with intact mitochondrial membrane 2. Stressed cells with dissipated membrane potential

hallmark of apoptosis or cell stress)

without Annexin V or 7-AAD staining

• Phospatidylserine translocation to the surface of early

3. Early apoptotic cells with dissipated membrane

apoptotic cells (measured by Annexin V binding)

potential and Annexin V binding

• Plasma membrane permeabilization or cell death

4. Late apoptotic cells or dead cells with dissipated

(measured by 7-AAD)

membrane potentials

2 µM Staurosporine 1.1% Red2 Fluoresecence (RD2-HLog)

Red2 Fluoresecence (RD2-HLog)

MitoSense Red

94.4%

104

103

102

101

100 0 10

0.75%

3.7% 101

102

103

3.7%

103

102

101

100 0 10

104

54.7%

50 µM CCCP 104

Red2 Fluoresecence (RD2-HLog)

Uninduced 104

14.6%

27.0% 101

Green Fluorescence (GRN-HLog)

102

103

0.04%

103

102

101

100 0 10

104

0.20%

93.2%

6.6% 101

Green Fluorescence (GRN-HLog)

102

103

104

Green Fluorescence (GRN-HLog)

Annexin V, CF488A

104

95.2%

104

0.3%

58.1%

104

0.08%

0.26%

2nd row: Cell death and mitochondrial membrane potential change

0.02%

102

101

Red2 Fluorescence (RD2-HLog)

Red2 Fluorescence (RD2-HLog)

Red2 Fluorescence (RD2-HLog)

MitoSense Red

Live Cells 103

103

Dead Cells

102

101

103

3rd row: Apoptosis and cell death.

102

101

Depolarized Cells 100 0 10

3.2%

1.3% 101

102

103

100 0 10

104

41.0%

0.8%

101

102

103

100 0 10

104

98.4%

1.3% 101

Red Fluoresecence (RED-HLog)

Red Fluorescence (RED-HLog)

102

103

104

Red Fluorescence (RED-HLog)

7-AAD 104

1.4%

103

102

101

100 0 10

95.2%

3.2% 101

102

103

Green Fluorescence (GRN-HLog)

104

0.06%

104

1.2% Red2 Fluorescence (RD2-HLog)

0.16%

Red2 Fluorescence (RD2-HLog)

Red2 Fluorescence (RD2-HLog)

7-AAD

104

103

102

101

100 0 10

70.5%

28.2% 101

102

103

Green Fluorescence (GRN-HLog)

104

 ot plots depicting Jurkat cells D stained using the MitoDamage kit. Jurkat cells uninduced, induced to apoptosis with 2 µM staurosporine or with 50 µM CCCP, then stained using the MitoDamage kit. Plots show the percentage of positive cells for:  1st row: Apoptosis (Annexin V binding) and mitochondrial membrane potential change

0.10%

1.2%

 ata reports that 2 µM D staurosporine induces apoptosis in Jurkat cells, and that 50 µM CCCP depolarizes the mitochondrial membrane, but neither condition is sufficient for cell membrane permeabilization and death.

103

102

101

100 0 10

93.9%

4.8% 101

102

103

104

Green Fluorescence (GRN-HLog)

Annexin V, CF488A

9

FlowCellect MitoPotential Red Kit

FlowCellect MitoStress Kit

Simultaneous analysis of mitochondrial membrane

Includes:

potential along with cell death provides key information

• MitoSOX™ Red, a live-cell-permeant, fluorogenic

for drug discovery, cancer and toxicology studies as well

dye which targets the mitochondria and reacts with

as disease-induced apoptosis. This kit uses MitoSense Red (a red laser-excitable dye) to monitor mitochondrial

superoxide radicals and fluoresces yellow/red • Annexin V conjugated to CF647 which binds to

membrane potential changes in early apoptosis, and

phosphatidylserine (PS) on the surface of apoptotic cells

7-AAD (a live-cell-impermeant DNA intercalator) to simultaneously monitor cell membrane permeability

The simultaneous use of these reagents enables researchers

changes in late apoptosis and necrotic cell death.

to obtain information on oxidative stress and apoptosis in one simple assay.

FlowCellect MitoLive Kit Includes:

FlowCellect Cytochrome c Kit

•  MitoSense Red, a fluorescent cationic dye that

Includes a directly labeled anti-Cytocrome c–FITC antibody

accumulates in the mitochondria and is responsive to

and Anti-IgG1-FITC isotype control. Viable or live cells will

mitochondrial potential changes

demonstrate higher levels of Cytochrome c staining while

•  Calcein acetoxymethylester (calcein-AM) a non-

apoptotic cells which have released their Cytochrome c

fluorescent, cell-permeant compound that is hydrolyzed

from the mitochondria to the cytoplasm will demonstrate

by intracellular esterases into the fluorescent anion

reduced staining intensity. The FlowCellect Cytochrome c

calcein and provides a measure of cellular vitality.

flow cytometry kit is a simple, gentle, and fast method to assess levels of Cytochrome c in mitochondria, providing a

The simultaneous use of the reagents enables researchers

valuable tool for assessing pro-apoptotic signaling and the

to obtain information on early and late apoptosis in one

efficacy of pro-apoptotic anti-cancer agents in cells.

simple assay.

FlowCellect Mitochondrial Kits Description

10

Qty

Catalog No.

FlowCellect MitoPotential Red Kit • Two dyes for measuring cell death and mitochondrial membrane potential • MitoSense Red (Red Laser) / 7-AAD (Blue Laser)

100 tests

FCCH100105

FlowCellect MitoDamage Kit • Three dyes to assess mitochondrial potential, stress, and cell death • Mitosense Red (Red) / Annexin V-CF488A (Blue) / 7-AAD (Blue Laser)

100 tests

FCCH100106

FlowCellect MitoLive Kit • Two dyes to measure mitochondrial health and cell vitality • Mitosense Red (Red) / Calcein-AM (Blue Laser)

100 tests

FCCH100107

FlowCellect MitoStress Kit • Understanding the regulation of apoptosis and oxidative stress. • MitoSox Red (Red) / Annexin V-CF647 (Blue Laser)

100 tests

FCCH100109

FlowCellect Cytochrome c Kit • Easy way to detect loss of mitochondrial Cytochrome c in cells • Anti-Cytochrome c-FITC (Blue) / Anti-IgG1-FITC (Blue Laser)

100 tests

FCCH100110

FlowCellect Oxidative Stress Characterization Kit • Detection of oxidative stress by flow cytometry • Anti-DNP-FITC (Blue Laser)

25 tests

FCCH025111

guava Mitochondrial Depolarization Assay Kit • Monitoring changes in mitochondrial membrane potential • JC-1 (Blue Laser) / 7-AAD (Blue Laser)

100 tests

4500-0250

Discover the power of flow cytometry for multiparametric apoptosis analysis. Apoptosis

Mid-Apoptosis Flow Cytometry Kits

Cells respond to specific apoptotic signals by initiating

Caspase activity is measured using a FLICA (fluorescent

intracellular processes that result in characteristic

labeled inhibitor of caspase) reagent, supplemented by

physiological changes. Among these changes are

a nuclear DNA stain 7-AAD, which evaluates membrane

externalization of phosphatidylserine to the cell surface,

integrity and cell viability. The assays are available in two

depolarization of mitochondrial membranes, cleavage

forms, sulforhodamine (SR) and carboxyfluorescein (FAM),

and degradation of specific cellular proteins, compaction

giving greater flexibility in assay design as well as the

and fragmentation of nuclear chromatin, loss of cell

capacity to multiplex caspase assays.

membrane integrity, and cellular shrinkage. Suppression or enhancement of apoptosis is known to cause or contribute

FLICA(-)7-AAD(-)

FLICA(+)7-AAD(-)

FLICA(+)7-AAD(+)

FLICA(-)7-AAD(+)

C+ C+ C+ C+

C+ C+ C+ PI+ C+ PI+

PI+ PI+ PI+ PI+

Early/Mid-stage (committed) apoptotic cells

Late stage apoptotic/dying cells

Dead cells

to diseases such as cancer and diabetes, making the apoptotic pathway a popular drug target. Because apoptosis is a dynamic event, and the time period during which cells exhibit apoptosis markers is variable and short, flow cytometry is an ideal technique for tracking

Live/Healthy (non-committed) cells

cells through apoptosis. Our easy-to-use kits enable you to examine cells at each of the various stages of apoptosis.

Early Apoptosis Flow Cytometry Kits

Late Apoptosis Flow Cytometry Kits

Two separate dyes identify a broad spectrum of

The guava TUNEL assay detects apoptosis-induced DNA

apoptotic and non-apoptotic cells: Annexin V binds to

fragmentation through a quantitative fluorescence assay.

phosphatidylserine on the external membrane of apoptotic

Terminal deoxynucleotidyl transferase (TdT) catalyzes the

cells, while 7-AAD permeates and stains DNA of late-stage

incorporation of bromo-deoxyuridine (BrdU) residues into

apoptotic and dead cells.

the fragmented nuclear DNA at the 3’-hydroxyl ends. A TRITC-conjugated anti-BrdU antibody then labels these

Advantages

DNA fragments. The assay distinguishes two populations:

• Two dye strategy : Detect various stages of apoptosis within a one assay

(TUNEL-positive).

non-apoptotic cells (TUNEL-negative) and apoptotic cells

• Mix-and-read assay: Get standardized results even with multiple users • All-in-one-kit : Spend less time before analysis • Compatible with pairing with FITC or PE probes or other probes in the green or yellow channels • Probe with other markers in green and yellow channels with the FlowCellect Mitochondrial kits PS

c

c

c

c

c+

PS

c+ PS

PS

c+ PS

Early Guava Nexin® Annexin Red MitoPotential Red MitoDamage MitoStress MitoLive Cytochrome c

A G...C C...G A...T

DNA strand breaks due to apoptosis

c+

c+

G

c+

G

G...C G...C A...T C...G

TdT + BrdUTP

G

G...C G...C A...T C...G

G...C G...C A...T C...G

A

A G...C C...G A...T

Add BrdUTP to 3’OH ends

TRITCAnti-BrdU

G...C C...G A...T

Antibody labeled break sites

PS

Mid Multicaspase Caspase 3/7, 8, 9 Dual Caspase

Late Guava TUNEL Assay

11

featured product FlowCellect Annexin Red Kit

Kit Component

In the early stages of apoptosis, phosphatidylserine molecules, which can bind to Annexin V, move from the

Laser

Buffer Pack

Annexin V-CF647

Red

7-AAD

Blue

10X Assay Buffer HSC

inner leaflet, to the outer leaflet of the plasma membrane. Apoptotic Cell Membranes

Healthy Cell Membranes

A rapid, sensitive, and convenient assay to monitor early

PS

and late apoptosis, the FlowCellect annexin red kit includes recombinant Annexin V conjugated to the red sensitive PS

dye CF647, and 7-AAD (a live cell-impermeant dye) to

PS PS

measure cell membrane integrity. After staining cells with

PS

PS

FlowCellect Annexin Red kit, three populations of cells can

= 7aad

PS

be identified in this assay:

Annexin CF647

PS = phosphatidylserine

• Non-apoptotic cells: Annexin V(-) and 7-AAD(-) • Early apoptotic cells: Annexin V(+) and 7-AAD(-)

PS

Two Dyes to distinguish early Apoptosis from later stages

• Late-apoptotic or dead cells: Annexin V (+) and 7-AAD(+) Uninduced

103

102

101

100 0 10

85%

11% 101

102

103

0.1 µM Staurosporine

104

2%

104

103

102

101

100 0 10

60%

36% 101

102

103

3 µM Staurosporine

104

4% Red2 Fluorescence (RD2-HLog)

2%

Red2 Fluorescence (RD2-HLog)

Red2 Fluorescence (RD2-HLog)

7-AAD

104

104

7%

103

102

101

100 0 10

5%

92% 101

102

103

Red2 Fluorescence (RED2-HLog)

Red2 Fluorescence (RED2-HLog)

Red2 Fluorescence (RED2-HLog)

A.

B.

C.

104

Annexin V

Dot plots depicting Jurkat cells stained using the FlowCellect Annexin Red kit. Jurkat cells were untreated (Plot A), treated with 0.1 µM (Plot B) or with 3 µM staurosporine (Plot C), and then stained using the FlowCelllect Annexin Red kit. The percentage of apoptotic cells increased from 36% to 92% in response to a 30-fold increase in staurosporine concentration; however, only a small fraction (< 10%) of the cells showed evidence of cell death.

Apoptosis Kits Description

Qty

Catalog No.

Early Apoptosis Kits FlowCellect Annexin Red Kit (Annexin V-CF647 Reagent/ 7-AAD)

100 tests

FCCH100108

Guava Nexin Reagent (Annexin V-PE/ 7-AAD)

100 tests

4500-0450

Guava MultiCaspase SR Kit (MultiCaspase SR reagent / 7-AAD)

100 tests

4500-0500

Guava Caspase 3/7 SR Kit (Caspase 3/7 SR reagent / 7-AAD)

100 tests

4500-0510

Guava MultiCaspase FAM Kit (Multicaspase FAM reagent / 7-AAD)

100 tests

4500-0530

Guava Caspase 3/7 FAM Kit (Caspase 3/7 FAM reagent / 7-AAD)

100 tests

4500-0540

100 tests

4500-0121

25 Tests

FCCS025108

Mid Apoptosis Kits For a complete listing visit: www.millipore.com/midapoptosis_kits

Late Apoptosis Kit Guava TUNEL Reagent Kit (Anti-BrdU -TRITC/ TdT Enzyme/ Br-dUTP)

Apoptosis Signaling Kit FlowCellect Bcl-2 Activation Dual Detection Kit (Anti-Bcl-2 -Alexa Fluor 488/ Anti-pBcl-2(Ser70) -PE) 12

Cell Signaling Discover the power of flow cytometry for multiparametric cell signaling research.  ual Detection FlowCellect Kits With Pairs of D Total and Phospho-Specific Antibodies

Advantages

such as apoptosis, cell differentiation, cell growth and cell proliferation, all of which have been extensively studied

EMD Millipore’s FlowCellect Dual Detection kits are a

in the process of developing therapies for various cancers

series of flow cytometry products which include a pair

• Ensures specific labeling of targets

and autoimmune disease. Cross-talk among signaling

of antibodies that bind to the same protein; one to

pathways adds an extra dimension of complexity when

detect total protein expression and another to detect

analyzing physiological consequences of a pathway of

the phosphorylated form of the same target. Using

interest. However, there are some key nodes at which

two parameter analysis, we can achieve target specific

multiple signals are integrated. Multiparametric flow

• Enables novice users to perform complex analysis

detection of phosphorylation and, by doing so, eliminate

cytometry provides researchers the power to monitor these

false positives while enhancing the signal to noise ratio.

Samples:

Signal transduction pathways lead to diverse outcomes,

intracellular ‘checkpoints’ simultaneously, enabling analysis of complicated cell events.

FlowCellect Cell Signaling Kits

F lowCellect Cell Signaling Kits With Directly Conjugated, Phospho-Specific Antibodies

Assay:

• Multiplex detection with no optimization required

• Designed to run 25 samples of human cells

Determine the effect of mechanical and chemical reagents

The study of cell signaling has been made easier by

that can induce DNA damage, discern multiple pathway

activation status-specific and phospho-specific antibodies.

activation and cross talk in a time-dependent manner,

Measuring the activity of cell signaling pathways by flow

or study the correlation between pathway activation and

cytometry delivers robust, high content information in

changes in cell function and health.

less time than traditional methods by analyzing multiple



parameters on hundreds of cells per second.

FlowCellect Kits: MAPK Pathway Description

FlowCellect PI3K/MAPK Dual Pathway Activation and Cancer Marker Detection Kit

Qty

Catalog No.

25 Tests

FCCS025100

25 Tests

FCCS025101

25 Tests

FCCS025106

25 Tests

FCCS025132

Anti-pErk1/2 (Thr202/Tyr204, Thr185/Tyr187)-PE Anti-phospho-Akt1/PKBa (Ser473), Alexa Fluor 488 Anti-KI-67- PerCP

FlowCellect EGFR/MAPK Pathway Activation Detection Kit Anti-pEGFR (Tyr1173), AlexaFluor 488 Anti-pERK1/2 (Thr202/Tyr204, Thr185/Tyr187)-PE

FlowCellect MAPK Activation Dual Detection Kit Anti-pERK1/2 (Thr202/Tyr204, Thr185/Tyr187)-PE Anti-ERK1/2-Alexa Fluor 647

FlowCellect p38 Stress Pathway Activation Detection Kit Anti-pP38 (Thr180/Tyr182), AlexaFluor 488 Anti-pATF2 (TThr69/71), AlexaFluor 647

FlowCellect Kits: EGFR Pathway Description

FlowCellect EGFR/MAPK Pathway Activation Detection Kit

Qty

Catalog No.

25 Tests

FCCS025101

25 Tests

FCCS025107

25 Tests

FCCS025111

Anti-pEGFR (Tyr1173), AlexaFluor 488 Anti-pERK1/2 (Thr202/Tyr204, Thr185/Tyr187)-PE

FlowCellect EGFR RTK Activation Dual Detection Kit Anti-pEGFR (Tyr1173), AlexaFluor 488 Anti-EGFR-PerCP

FlowCellect EGFR/STAT3 Pathway Activation Detection Kit Anti-pEGFR (Tyr1173), Alexa Fluor 488 Anti-pSTAT3 (Tyr705), Alexa Fluor 647

13

featured product FlowCellect PI3K/MAPK Dual Pathway Activation and Cancer Marker Detection Kit

IGF-1

Wortmannin or LY294002

PMA

Three antibodies to study cross-talk between the PI3K and MAPK pathways. The kit uses directly labeled antibodies against phospho-Akt1/PKBa(Ser473)-AlexaFluor 488 and

P

P

P

P

Anti-phospho-Erk1/2 (Thr202/Tyr204, Thr185/Tyr187)-PE to analyze signaling activation and cross talk, plus Ki-67

Shc

Grb2

SOS

PI3-Kinase

marker-PerCP marker to identify the proliferative fraction. Together, the antibody trio makes it easy to evaluate the

p85

GTP

p110

RAS

role these signaling pathways play in proliferation and

GDP

differentiation. The kit also includes Cell Cycle Stop™

P

fixation reagent to improve Ki-67 detection. Although

Akt

Raf-1

Ki-67 is present throughout most of the cell cycle, it is difficult to detect by flow cytometry except during M phase. Cell cycle stop reagent arrests the cycle at the

MEK1/2

M phase, making it possible to accurately detect Ki-67 Inhibiting

expression and discern the biological effects of the PI3K/

Activating

P

MAPK cross talk.

ERK1/2

Proliferation/ Survival

Growth/ Differentiation

Ki 67

A Cancer Proliferative Marker

Untreated

Insulin Treated

PMA Treated C. HEK203 - 3 Minute Stimulation

103

103

103

102

101

101 102

103

100 0 10 101

104

pAKT-Alexa 488

pERK

101 102

103

104

pAKT-Alexa 488

B. 5 Minute Stimulation

102

104

103

103

103

pAKT-Alexa 488

104

104

102

101 102

103

Untreated 100 nM insulin 3 min 100 µg/mL PMA 3 min

pAkt

pErk

Ki67

D. HEK203 - 5 Minute Stimulation 104

102

101

102

100 90 80 70 60 50 40 30 20 10 0

pAKT-Alexa 488

pERK-PE

pERK-PE

103

100 0 10 101

100 0 10 101

pERK-PE

100 0 10 101

104

102

% of cells

102

pERK-PE

104

pERK-PE

104

pERK-PE

104

% of cells

A. 3 Minute Stimulation

101

100 0 10 101

102

103

pAKT-Alexa 488

104

100 0 10 101

102

103

pAKT-Alexa 488

104

100 90 80 70 60 50 40 30 20 10 0

Untreated 100 nM insulin 3 min 100 µg/mL PMA 3 min

pAkt

pErk

Ki67

pAKT

14

The cross-talk between the PI3K/Akt and MAPK signaling pathways is evident downstream of IGF in HEK293 cells. Cells are stimulated at 3 minutes and 5 minutes by Insulin and PMA as shown in A and B, respectively. As previously indicated, although Insulin initially activates both pathways independently, the activation of the PI3K pathway indicated by the phosphorylation of Akt and ERK1/2 inhibits the activation of ERK. The dot plots illustrate the cross-talk that exists between these two signaling pathways. This is demonstrated by the sharp decrease in ERK phosphorylation between 3 minutes and 5 minutes (see bar graphs; C, D).

featured product FlowCellect Kit EGFR RTK Activation Dual Detection Kit EGF EGFR

EGF pathway activation through EGFR plays a key role in the regulation of essential cellular processes, and abnormality of EGF signaling is linked to cancer. EMD

P

Millipore’s FlowCellect EGFR RTK Activation Dual Detection

P

kit includes two directly conjugated antibodies, a phosphospecific Anti-phospho-EGFR (Tyr1173)-Alexa Fluor 488

JAK

PI3K

Ras

and an Anti-EGFR-PerCP conjugated antibody to measure total levels of EGFR. This two color flow cytometry kit is STAT

designed to detect the extent of EGF pathway activation

AKT

by measuring the EGFR phosphorylation in relative to the

ERK

total EGFR expression in any given cell population. By

mTOR

doing such, the levels of both the total and phosphorylated protein can be measured simultaneously in the same cell, resulting in a normalized and accurate measurement of EGFR activation after stimulation. Moreover, simultaneous measurement of both total and phospho-EGFR confirms target specificity of the phosphorylation event. Together, a total and phospho antibody duo performed in multiplex provides an enhanced and more reliable detection of the phospho:total ratio within a mixed population.

A. Isotype Control (No EGF Stimulation)

B. Total and pEGFR (No EGF Stimulation)

104

104

98%

1.5% pEGFR-Alexa Fluor 488

pEGFR-Alexa Fluor 488

0% 103

102

101

0.5%

100 100

101

102

103

0.5%

5.4%

103

102

101

88.6%

5.5%

100 100

104

101

pEGFR

EGFR-PerCP

104

0.3%

103

102

101

99% 100 100

0.7% 101

102

104

D. Total and pEGFR (EGF Stimulation)

pEGFR-Alexa Fluor 488

pEGFR-Alexa Fluor 488

0%

103

EGFR-PerCP

C. Isotype Control (EGF Stimulation) 104

102

103

EGFR-PerCP

98.2

103 102

101 100 100

104

0.6%

1.0%

0.2% 101

102

103

104

Dual Parameter Analysis of Total and Phospho EGFR on A431Cells As illustrated in dot plots A and B, untreated A431cells stained with an isotype control (A) and both pEGFR-Alexa Fluor 488 and Anti-EGFR-PerCP (B), respectively, only indicated EGFR expression on total EGFR as noted by 89% of cells. However, once A431cells were stimulated with 100 ng/ mL EGF, simultaneous measurement of both total and phospho EGFR confirms target specificity of the phosphorylation event as indicated by the double positive cell population (D) as indicated by the 5% to 98% increase of double positive staining. A431 stimulated cells showed no activity when stained with an isotype control (C).

EGFR-PerCP

Total EGFR

15

FlowCellect Cell Signaling Kits Description

Qty

Catalog No.

PI3/ Akt/ m-TOR Pathway FlowCellect PI3K/MAPK Dual Pathway Activation and Cancer Marker Detection Kit

25 Tests

FCCS025100

25 Tests

FCCS025105

25 Tests

FCCS025210

25 Tests

FCCS025111

25 Tests

FCCS025550

25 Tests

FCCS025142

25 Tests

FCCS025143

25 Tests

FCCS025154

25 Tests

FCCS025145

25 Tests

FCCS025108

Anti-phospho-Erk1/2 (Thr202/Tyr204, Thr185/Tyr187)-PE / Anti-p-Akt1/PKBa (Ser473)- Alexa Fluor 488 / Anti-KI-67- PerCP

FlowCellect PI3K Activation Dual Detection Kit Anti-phospho-Akt (Ser473) Alexa Fluor 488 / Anti-Akt/PKB-Alexa Fluor 647

FlowCellect PI3K-mTOR Signaling Cascade Mapping Kit Anti-p-Ribosomal Protein S6 (Ser235)-PerCP / Anti-p-Akt1/PKBa (Ser473)-Alexa Fluor 488

Jak/ STAT Pathway FlowCellect EGFR/STAT3 Pathway Activation Detection Kit Anti-pEGFR (Tyr1173), Alexa Fluor 488 / Anti-pSTAT3 (Tyr705), Alexa Fluor 647

FlowCellect Multi-STAT Activation Profiling Kit Anti-pSTAT1 (Tyr701)-PerCP / Anti-pSTAT3 (Tyr705)-Alexa 488 / Anti-pSTAT5A/5B (Tyr694/Tyr699)-PE

FlowCellect STAT1 Activation Dual Detection Kit Anti-p-STAT1(Tyr701) Alexa Fluor 488 / Anti-STAT1-PerCP

FlowCellect STAT3 Activation Dual Detection Kit Anti-p-STAT3 (Tyr705) Alexa Fluor 647 / Anti-STAT3-Alexa Fluor 488

Multiple Pathway FlowCellect Src Activation Dual Detection Kit Anti-pSrc (Tyr416)-Alexa Fluor 488 Anti-Src-Alexa Fluor 647

FlowCellect PLC-γ1 Activation Dual Detection Kit Anti-pPLC-γ1 (Tyr783) Alexa Fluor 488 Anti-PLC-γ1-PE

Apoptosis Signaling Pathway FlowCellect Bcl-2 Activation Dual Detection Kit Anti-Bcl-2 Alexa Fluor 488 Antibody Anti-pBcl-2(Ser70) PE

F lowCellect Chemokine Receptor Surface Expression Quantification Kits EMD Millipore offers 11 GPCR surface identification flow

Advantages

cytometry kits. Flow cytometry provides high quality,

Assay:

reproducible data in far less time than traditional methods

• Includes pharmacologically characterized positive and negative control cells

for chemokine receptor research and avoids the hazard and expense of radioligand binding assays.

• Same accuracy as radioactive assays, without the hazards.

Our FlowCellect GPCR identification kits can be used to

• Ability to identify high, medium, and low expressing cell cultures during the clonal selection process.

identify and quantify GPCRs on the surface of any cells. antibody validated for flow cytometry. Also included are

• High reproducible results obtained by using EMD Millipore’s well-characterized ChemiScreen™ GPCR cell lines as assay controls

positive and negative control cells with well-characterized

Samples:

receptor expression levels for the purposes of quantitative

• Sufficient reagents to run 100 samples of human cells.

The kits detect GPCR expression using a GPCR-specific

extrapolation. Chemokine Receptor Kits Description

16

Qty

Catalog No.

FlowCellect Chemokine Receptor CCR2B Surface Expression Quantification Kit

100 tests

FCCR200411

FlowCellect Chemokine Receptor CCR4 Surface Expression Quantification Kit

100 tests

FCCR400413

Chemokine Receptor CCR6 Surface Expression Quantification Kit

100 tests

FCCR600414

FlowCellect Chemokine Receptor CCR7 Surface Expression Quantification Kit

100 tests

FCCR700415

For a complete listing of kits visit: millipore.com/flowcytometry.

Stem Cells Discover the power of flow cytometry for multiparametric stem cell analysis. Because flow cytometry has the power to characterize

Embryonic Stem Cell Markers

subpopulations of cells within heterogenous cell mixtures; it is widely used for studying both embryonic stem cells

Differentiated cells

(ESCs) and induced pluripotent stem (iPS) cells. Flow

Oct-4SSEA-4TRA1-60HESCASSEA-1+/-

cytometry enables researchers to evaluate percentages of cells expressing specific markers, to determine culture quality, and to track gene expression changes during a differentiation protocol.

FlowCellect Stem Cell Characterization Kits

Stem cell

(hESC, mESC) Oct-4+ SSEA-4+ (human) TRA1-60+ HESCA+ Nanog+ SSEA-1+ (mouse)

Committed cell (ENStem-A) Oct-4-/+ SSEA-4TRA1-60HESCA-

EMD Millipore’s FlowCellect stem cell characterization kits are designed to provide rapid, sensitive assessments of embryonic and neural stem cell phenotypes at various

Advantages

stages. The kits use three parameters for accurate

Assay:

identification, enabling the research to “triangulate” cellular phenotypes with two complementary positive

• All optimized, fluorescently-labeled flow cytometry antibodies and buffers included

markers and one negative marker. The negative antibody

• Highly reproducible results

also serves as a stage- and species-specific or lineage-

• Validated for flow cytometry, immunocytochemistry, and Western blotting

specific marker for differentiated cells.

Stem Cell FlowCellect Kits Description

Qty

Catalog No.

Human FlowCellect Human ESC (Oct4) Nuclear Marker Characterization Kit hOCT4-Alexa 488 / SSEA4-PE / SSEA1-PE/CY5

25 Test

FCHEC25102

FlowCellect Human ESC (HESCA-1) Surface Marker Characterization Kit

25 Tests

FCHEC25104

25 Tests

FCHEC25106

25 Tests

FCMEC25110

25 Tests

FCRNC25112

HESCA1-FITC / SSEA4-PE / SSEA1-PE/CY5

FlowCellect Human ESC (TRA-1-60) Surface Marker Characterization Kit TRA-1-60-FITC / SSEA4-PE / SSEA1-PE/CY5

Mouse FlowCellect Mouse ESC (Oct4) Nuclear Marker Characterization Kit mOCT4-Alexa 488 / SSEA4-PE / SSEA1-PE/CY5

Rodent FlowCellect Rodent NSC Characterization Kit (Neuronal Differentiation) Sox-2 FITC / Nestin-PE / Beta-III-Tubulin-PE/CY5

17

featured products FlowCellect Human Embryonic Stem Cell Characterization Kit

10e4

and SSEA4 but not SSEA1. This quick test will enable

expression of hOCT4, SSEA4 and SSEA1. The percentage of

researchers to determine the multipotency of their cells in

undifferentiated human embryonic stem cells in culture is

culture as well as see changes in marker expression during

reflected in the percentage cells that express both hOCT4

a differentiation protocol.

0%

0.1%

B.

85.5%

0.1%

10e4

C.

10e3

hOCT4-Alexa 488

10e3

SSEA1-PE/CY5

10e4

10e2

10e2

10e1

10e1

4.9%

10e0

10e0

95% 10e1

10e2

SSEA4-PE

10e3

85.5%

10e2

10e1

14.4%

10e0

10e0

10e4

0.1%

10e3

hOCT4-Alexa 488

A.

The kit is provides a quick, easy way to track surface marker

0% 10e1

10e2

SSEA1-PE/CY5

10e3

4.8%

10e0 10e0

10e4

9.6% 10e1

10e2

SSEA4-PE

10e3

10e4

Representative data of H1 human embryonic stem cells stained with hOCT4-Alexa 488, SSFA4-PE and SSFA1-PF/CY5

FlowCellect Human Embryonic Stem Cell HESCA-1 Surface Marker Characterization Kit This kit provides an easier and quick way to track surface

HESCA-1 and SSEA4 and not SSEA1. This quick test will

marker expression of HESCA-1, SSEA4 and SSEA1.

enable researchers to test the quality of their cells in

The kit will also help to determine the percentage of

culture as well as see changes in marker expression during

undifferentiated human embryonic stem cells in culture

a differentiation protocol.

by determining the percentage of cells that express both A.

B.

10e4

10e3

10e2

10e2

10e1

10e1

10e0 10e0

10e1

10e2

SSEA4-PE

10e3

10e4

10e0 10e0

10e4

10e3

SSEA1-PE Cy5

HESCA-FITC

10e3

HESCA-FITC

C.

10e4

10e2

10e1

10e1

10e2

SSEA1-PE Cy5

10e3

10e4

10e0 10e0

10e1

10e2

SSEA4-PE

Representative data of H1 human embryonic stem cells stained with HESCA-1-FITC, SSEA4-PE and SSEA1-PE/CY5.

18

10e3

10e4

Immunology Discover the power of flow cytometry for multiparametric analysis of the immune system. FlowCellect Immunology Kits

The immune system, which mediates the body’s response to the introduction of foreign material, is made up

Each kit includes multiple optimized fluorescent labeled

of multiple cell types collectively called lymphocytes.

antibodies and all buffers necessary for cell preparation and

Lymphocyte subtypes include B cells (which secrete

analysis. Detailed assay instructions are included to assist in

antibodies), cytotoxic T cells, helper T cells (which secrete

analysis and to ensure that the correct cell concentration is

cytokines), and natural killer (NK) cells. Characterization of

obtained during acquisition of sample data.

lymphocyte subtypes and cytokine signaling is essential for understanding the complex nature of the immune system.

Advantages

Activation by antigens, suppression of normal immune

Assay:

activation, and disease states can affect the phenotypes

• Multiplex detection with no optimization required

of lymphocytes. Multiparameteric phenotypic analysis

• Highly reproducible

by flow cytometry allows researchers to distinguish one

• Minimal assay development needed

subpopulation of cells within a heterogeneous mixture, and thereby enables the study the dynamics of immune

• All optimized flow cytometry antibodies and buffers included

signaling in intact cells.

• Enables novice users to perform complex analysis Samples: • Designed to run 25 tests (FlowCellect kits)

CD4+ T-Cell Differentiation

• Designed to run 100 tests (guava kit)

IFNγ IL-12R

IL-2

TH 1

LTα

T-bet STAT4 STAT1

IL-4 IL-4R

TH 2

IL-13

CCR7lo

IL-25

DC

CD80 pMHCII

TCR CD28

B ZONE

IL4 IL2 IL-

CD28

CXCR5hi

33

IL-12 IFNγ IL-18

GATA3 STAT6 STAT5a

High TCR Binding

IL-5

IL-21R

TH IL-6

naive

IL-21

TFH Bcl-6 STAT3

IL-21 IL-17

Medium TCR Binding CD62Llo

CCR7lo

EMIGRANT

CD85 6

ILβ

F TG

TGFβ

IL23

IL-2

IL-23R

IL-2R

RORγt STAT3

TREG Foxp3 STAT5

TH 17

IL-17 IL-17F

Low TCR Binding CD62Lhi

CCR7hi

T ZONE

IL-22 IL-21

TGFβ IL-10 IL-35

 D4+ T Cell Differentiation. Schematic diagram of CD4+ T-cell differentiation. Five different types of CD4+ T-cells can develop from C a common naïve precursor depending on the cytokine environment and interaction with dendritic cells (DC).

19

featured product FlowCellect Mouse Th1/Th17 Intracellular Cytokine Kit 9000

9000 3.7%

7000

7000

5000 3000 1000 0 100

101

102

FVD-660

103

104 3.7%

104

102 101 102

36.1% 103 104

to detect IFN-g and IL-17 expression in mouse Th1 and Th17 CD4+ T-cells. This kit contains an anti-IFN-g antibody conjugated to PE, an anti-IL17 antibody conjugated FITC

3000 1000 4.2% 0 100 101

and an anti-CD4 antibody conjugated to PerCP. The kit also 36.1%

102

FVD-660

103

104 1.3%

Il-17-FITC

IFNγ-PE

103

Kit is designed to enable a researcher a quick and easy way

56.0%

5000

56.0%

4.2% 100 0 10 101

EMD Millipore’s FlowCellect Mouse Th1/Th17 Identification

Th-17 Cells

Side Scatter

Side Scatter

Th-1 Cells

104

32.5%

103

includes optimized a protein transport inhibitor and buffers to aid in identifying Th1 and Th17 CD4+ T-cell subsets in ex vivo lymphocyte populations or to monitor Th1 and Th17 CD4+ Tcell differentiation in culture. In addition, EMD Millipore has added a fixable viability dye that eliminates

102

false positive data resulting from cytokine staining on dead

101

cells that can occur in long term cultures. The kit contains

7.4% 100 0 10 101

CD4-PerCP

102

58.8% 103 104

CD4-PerCP

sufficient reagents for 25 3-color tests. Detailed assay instructions are included to assist in sample preparation and to ensure that the correct cell concentration is obtained during acquisition of sample data. This kit is not designed to be used in the analysis of whole spleen cells in SJL mice.

Helper T-cell Kits Mouse Description

FlowCellect Mouse Th1 Intracellular Cytokine Kit

Qty

Catalog No.

25 Tests

FCIM025123

25 Tests

FCIM025124

25 Tests

FCIM025125

25 Tests

FCIM025137

25 Tests

FCIM025138

Anti-CD4 clone GK1.5-PerCP /Anti-IFNγ, clone XMG1.2-PE A quick and easy way to detect IFNγ-γexpression in mouse Th1 CD4+ T cell

FlowCellect Mouse Th2 Intracellular Cytokine Kit Anti-CD4-PerCP clone GK1.5/ Anti-IL-4, clone 11B11-PE A quick and easy way to detect IL-4 expression in mouse TH2 CD4+ T-cells

FlowCellect Mouse Th17 Intracellular Cytokine Kit Anti-CD4-PerCP clone GK1.5/ Anti-IL-17-, clone TC11-18H10-FITC A quick and easy way to detect IL-17 expression in mouse TH17 CD4+ T-cells.

FlowCellect Mouse Th1/Th2 Intracellular Cytokine Kit 20X Anti-CD4 clone GK1.5-PerCP / Anti-IL-4, clone 11B11-PE/ Anti-IFN-γ, clone XMG1.2-PE A quick and easy way to detect IFN-γ and IL-4 expression in mouse Th1 and Th2 CD4+ T-cells.

FlowCellect Mouse Th1/Th17 Intracellular Cytokine Kit Anti-CD4-PerCP clone GK1.5/ Anti-IL-17, clone TC11-18H10-FITC/ Anti-IFNγ, clone XMG1.2-PE A quick and easy way to detect IFN-γ and IL-17 expression in mouse TH1 and TH17 CD4+ T-cells.

Regulatory T-cell Kits Human Description

FlowCellect Human FOXP3 Treg Characterization Kit

Qty

Catalog No.

25 Tests

FCIM025118

100 Tests

FCIM100158

Anti-CD3-PE/Cy5/ Anti-CD4-FITC/ Anti-FOXP3-Alexa Fluor 647

FlowCellect Human CD4/CD8 T Cell Kit Anti-CD8-FITC, CD4-PE, CD3-PECY5 of cocktail

Mouse Description

FlowCellect Mouse FOXP3 Treg Identification Kit

Qty

Catalog No.

25 Tests

FCIM025126

25 Tests

FCIM025168

Anti-CD4 clone GK1.5-PerCP/ Anti-FOXP3, clone 3G3- AlexaFluor 647

FlowCellect Mouse Viable Treg Characterization Kit Anti-CD4-PerCP/Cy5.5 / Anti-CD25-PE/ Anti-Foxp3-Alexa Flour 488

20

featured product FlowCellect Human Memory B cell Identification Kit We have developed a multi-parameter flow cytometry

bench top flow cytometers. FlowCellect kits can be used on

assay for monitoring human memory B cell function. EMD

any flow cytometer following the same protocol providing

Millipore’s FlowCellect Human Memory B Cell Identification

researchers a reliable and fully validated solution to study

Kit includes three directly conjugated antibodies: Anti-

and identify human B cell function right in the comfort

Human CD5-FITC, Anti-Human CD19-APC, and Anti-

of their own lab. All three antibodies provided in the kit

Human CD27-PE, along with optimized assay buffers to

are carefully titrated and optimized together to ensure

provide researchers the ability to phenotypically distinguish

maximal performance when run in multiplex, alleviating

cell types. All FlowCellect kits are optimized on guava

the need for any additional optimization.

A.

104

B.

104

3000

102

101

Lymphocyte 0 1000

3000

5000 7000

9000

100 0 10

Memory B-cell

103

PE-CD27

5000

1000 0

CD5+B-cell

103

7000

FITC-CD5

Side Scatter

9000

C.

102

101

101

Forward Scatter

102

103

104

APC-CD19

100 0 10

102

101

103

104

APC-CD19

Isolation and Identification of Human Memory B cells from human PBMCs Human memory B cells are phenotypically identified by using specific human CD markers: Human CD5, CD19, and CD27. CD27 has been identified as the key marker for identifying memory B cells. In (A), lymphocyte populations are gated, and B cells are shown using bivariate analysis plotting CD5+CD19+ (B). Memory B cell subsets are also identified by using a CD19+CD27+ (C). In healthy patients, memory B cells represent approximately 30-60% of the B-cell pool. B-cell subpopulations are defective in patients suffering from immuno-deficiency disorders, showing a reduced number of circulating CD19+CD27+ memory B cells.

B-cell Human: B-cell Description

FlowCellect Human Memory B Cell Identification Kit

Qty

Catalog No.

25 test

FCIM025159

100 Tests

FCCH100137

Anti-CD5 clone DK23-FITC / Anti-CD19 clone HD37-APC / Anti-CD27 clone M-T271-PE

FlowCellect Human B Cell FAS Kit CD19-FITC, CD45-PerCP of Cocktail, Anti-Human CD95(FAS)-PE / Isotype control mouse IgG1-PE

Mouse: B-cell Description

FlowCellect Mouse Breg Identification Kit

Qty

25 Tests

Catalog No.

FCIM025154

Anti-Human CD5, clone 53-7.3-APC / Anti-Mouse CD19, clone 1D3-FITC / Anti-Mouse CD1d, clone 1B1-PE / Anti-Mouse CD16/CD32, clone 93 Purified

T-cell Signaling Kit Human: T-cell Signaling Description

FlowCellect Human Lymphocyte ZAP-70 Characterization Kit

Qty

25 Tests

Catalog No.

FCIM025122

Anti-CD5-FITC/ Anti-CD19-APC/ Anti-ZAP-70-PE

21

featured product

Immune Cell Health (Apoptosis) FlowCellect Human T-Cell MitoDamage Kit

dissipated mitochondrial membrane potential (3) CD8

EMD Millipore’s FlowCellect T Human T cell MitoDamage Kit

cytotoxic T Cells and the % of these cells that show intact

includes (1) Antibody Cocktail containing CD3-PECy5, CD4-

mitochondrial membrane potential change and (4) CD8

PE and CD8-FITC antibodies (2) MitoSense Red (1,1’,3,3,3’,3’

Cytotoxic T cells and % of these cells that demonstrate

-Hexamethylindodicarbocyanine iodide), a fluorescent

dissipated membrane potential. The kit thus provides a

cationic dye that accumulates in the mitochondria and is

complete picture of T cell mitochondrial perturbation

responsive to mitochondrial potential changes and (3) 1X

status and its response for inducer treatment conditions

Assay buffer BA solution to perform the assays. The kit

or diseases. The entire assay can be performed in 30 min

can thus distinguish multiple populations (1) CD4 T Helper

a simple no wash manner without loss of apoptotic cells

cells and % of these cells which show intact mitochondrial

when using PBMC’s.

membrane potential (2) % of CD4 T Helper cells with Untreated

Diamide

SSC

B

CD8+ FITC

2

MitoSense Red

CD8 FITC

1

CD8+ FITC

A

MitoSense Red

CD4-PE

D

CD4-PE

CD3-PECy5

CD4-PE

C

MitoSense Red

MitoSense Red

Mitochondrial membrane depolarization in apoptotic CD4 and CD8 T cells PBMCs were treated overnight with and without 50 µM diamide and analyzed with the FlowCellect T Cell MitoDamage Kit. Plots show the percentage of positive cells for CD3, CD4 and CD8 subpopulations (1, 2) and the mitochondrial potential status of CD4 and CD8 T cell populations for untreated (A, C) and treated samples (B, D). Cells with intact mitochondrial potential exhibit higher Red2 or MitoSense red fluorescence (as in untreated control), while cells with depolarized mitochondria demonstrate a downward shift in their mitochondrial potential.

Human Description

FlowCellect Human T Cell Apoptosis Kit

Qty

Catalog No.

100 Tests

FCCH100138

100 Tests

FCCH100139

100 Tests

FCCH100140

100 Tests

FCCH100141

100 Tests

FCCH100154

100 Tests

FCCH100155

100 Tests

FCCH100156

100 Tests

FCCH100157

100 Tests

FCCH100137

100 Tests

4500-0230

(CD8-FITC, CD4-PE, CD3-PECy5 of cocktail/ Annexin V, CF647 Reagent)

FlowCellect Human T Cell MitoDamage Kit (CD8-FITC, CD4-PE, CD3-PECy5 of cocktail/ MitoSense Red)

FlowCellect Human CD8 T Cell FAS Kit (CD8-FITC, CD3-PECY5 of Cocktail/ Anti-Human CD95(FAS)-PE/ Isotype control mouse IgG1-PE)

FlowCellect Human T Cell Activation Kit (CD4-FITC, CD69-PE, CD3-PECY5, CD8-APC of cocktail)

FlowCellect Human CD4 T Cell FAS Kit (CD4-FITC, CD3-PECY5 of cocktail/ Anti-Human CD95(FAS)-PE / Isotype control mouse IgG1-PE)

FlowCellect Human T Cell Caspase 8 Kit (CD4-PE, CD3-PECy5, CD8-APC of cocktail/ Caspase 8 FAM)

FlowCellect Human T Cell Caspase 9 Kit (CD4-PE, CD3-PECy5, CD8-APC of cocktail/ Caspase 9 FAM)

FlowCellect Human T Cell Caspase 3/7 Kit (CD4-PE, CD3-PECy5, CD8-APC of cocktail/ Caspase 3/7 FAM)

FlowCellect Human B Cell FAS Kit (CD19-FITC, CD45-PerCP Cocktail, Anti-Human CD95(FAS)-PE/ Isotype control mouse IgG1-PE)

Guava Cell Toxicity Kit (Guava CFSE/ CellToxicity 7-AAD)

22

Milli-Mark Conjugated Antibodies 200

Advance your flow cytometry analysis with EMD

Stem Cell Fixed and permeabilized 2102Ep embryonal carcinoma cells were stained with MilliMark anti-hOct4-Alexa Fluor 488 (FCMAB113A4, green histogram) or IgG-Alexa Fluor 488 (grey histogram) at 1:100 for 1 hour and then analyzed by flow cytometry.

180

Millipore’s growing selection of directly conjugated

160 140

flow cytometry solution including instruments, software,

120

service, and reagents, Milli-Mark fluorescently-labeled

Count

primary antibodies. As a part of our complete benchtop

100 80

antibodies are specifically designed, optimized and

60

validated for flow cytometry applications.

40 20 0 10e0

10e1

10e2

10e3

10e4

hOCT4-Alexa Fluor 488

Milli-Mark: Stem Cell Description

Reactivity

Host

Anti-hOCT4-Alexa Fluor 488

Human

Mouse

100 tests

Qty

FCMAB113A4

Catalog No.

Mouse Anti-SSEA-4-PE

Human

Mouse

100 tests

FCMAB116P

Anti-Sox2-FITC

Human

Mouse

100 tests

FCMAB112F

Mouse Rat Anti-TRA160-FITC

Human

Mouse

100 tests

FCMAB115F

Anti-HESCA-1-FITC

Human

Mouse

100 tests

FCMAB111F

Mouse

100 tests

FCMAB117P

Mouse Anti-SSEA-1-PE

Human Mouse Rat

Anti-SSEA3 Alexa 488, clone MC-631

Human

Rat

100 tests

FCMAB141A4

Anti-Human Nuclei -FITC, clone 3E1.3

Human

Mouse

100 tests

FCMAB157F

Anti-BMP-7-FITC, clone 2A10

Human

Mouse

100 tests

FCMAB135F

Anti-mOCT4-Alexa Fluor 488, clone 7F9.2

Human

Mouse

100 tests

FCMAB124A4

Mouse Anti-TRA-1-81-FITC,clone TRA-1-81

Human

Mouse

100 tests

FCMAB132F

Anti-TRA-2-49-FITC, clone TRA-2-49/6E

Human

Mouse

100 tests

FCMAB133F

Anti-SRF-FITC, clone 1E1

Human

Mouse

100 tests

FCMAB137F

Description

Reactivity

Host

Qty

Catalog No.

Anti-phospho-Bcl-2 (Ser70)-PE, clone 69-10C-2-10C-18

Human

Rabbit

100 tests

FCMAB140P

Rat-a-Caspase-2-FITC

Human

Rat

100 tests

FCMAB158F

Milli-Mark: Apoptosis and Cancer

23

200 180 160 140

Count

Epigenetics & Gene Regulation HeLa cells were stained with either Milli-Mark anti-Y14FITC, clone 4C4 (FCMAB151F, green histogram) or with IgG2b isotype control antibody (grey histogram) and analyzed using flow cytometry.

120 100 80 60 40 20 0 10e0

10e1

10e2

10e3

10e4

Green Fluorescence

Milli-Mark: Epigenetics and Gene Regulation Description

Reactivity

Host

Qty

Catalog No.

Anti-MAD2A-FITC, clone 17D10

Human

Mouse

100 tests

FCMAB150F

Anti-Y14-FITC, clone 4C4

Human

Mouse

100 tests

FCMAB151F

Anti-FXR1-FITC, clone 6BG10

Human

Mouse

100 tests

FCMAB152F

Anti-SMN-FITC, clone 2B1

Human

Mouse

100 tests

FCMAB153F

Anti-CAF1 p150-FITC, clone SS1, 1-3

Human

Mouse

100 tests

FCMAB145F

Anti-TBX21/T-Bet-FITC

Human

Mouse

100 tests

FCABS131F

Anti-RPA2 p34 -FITC, clone RPA20 1-46

Human

Mouse

100 tests

FCMAB143F

Anti-RPA1 p70 -FITC, clone RPA9, 1-30

Human

Mouse

100 tests

FCMAB144F

Anti-BMI1-FITC, clone AF27

Human

Mouse

100 tests

FCMAB149F

Anti-RBMS1-FITC, clone 4D11

Human

Mouse

100 tests

FCMAB123F

Anti-c-Jun-FITC, clone 6E4

Human

Mouse

100 tests

FCMAB122F

Mouse

100 tests

FCMAB125F

Mouse Rat Anti-RBMS1-FITC, clone 4D11

Human Mouse

Anti-BrdU-Alexa 488

Human

Mouse

100 tests

FCMAB101A4

Phospho-ATM (Ser1981) - PE

Human

Mouse

100 tests

FCMAB110P

Mouse Rat Phospho H3 (Ser10)- Alexa 488

Human

Mouse

100 tests

FCMAB104A4

Phospho-SMC1 (Ser957) - Alexa 488

Human

Mouse

100 tests

FCMAB108A4

Mouse

100 tests

FCMAB102P

Bovine Xenopus Anti-Cyclin B1 - PE

Human Mouse

24

Milli-Mark: Inflammation/Immunology Description

Reactivity

Host

Qty

Catalog No.

Anti-CD1a-FITC, clone NA1/34

Human

Mouse

100 tests

FCMAB166F

Anti-CD2-FITC, clone MT910

Human

Mouse

100 tests

FCMAB167F

Anti-CD3-PECy5, clone UCHT1

Human

Mouse

100 tests

FCMAB169C5

Anti-CD4-FITC, clone MT310

Human

Mouse

100 tests

FCMAB170F

Anti-CD8-FITC, clone DK25

Human

Mouse

100 tests

FCMAB176F

Anti-CD11c-FITC, clone KB90

Human

Mouse

100 tests

FCMAB179F

Anti-CD13-FITC, clone WM-47

Human

Mouse

100 tests

FCMAB180F

Anti-CD14-FITC, clone TUK4

Human

Mouse

100 tests

FCMAB181F

Anti-CD16-FITC, clone DJ130c

Human

Mouse

100 tests

FCMAB183F

Anti-CD19-APC, clone HD37

Human

Mouse

100 tests

FCMAB185AP

Anti-CD27-FITC, clone M-T271

Human

Mouse

100 tests

FCMAB191F

Anti-CD45-PE, clone F10-89.4

Human

Mouse

100 tests

FCMAB118P

Anti-CD45RA-FITC, clone MEM 56

Human

Mouse

100 tests

FCMAB126F

Mouse Anti-CD56-PE, clone MOC-1

Human

Mouse Mouse

100 tests

FCMAB200P

Anti-CD57-FITC, clone TB01

Human

Mouse Mouse

100 tests

FCMAB201F

Description

Reactivity

Host

Anti-EGFR-PerCP, clone LA22

Human

Mouse

100 tests

FCMAB129CP

Anti-mTOR-FITC, clone 2ID8.2

Human

Mouse

100 tests

FCMAB154F

Anti-Ras-FITC, clone RAS10

Human

Mouse

100 tests

FCMAB148F

Anti-GbL/mLST8, clone 3E1.2, FITC Conjugate

Human

Mouse

100 tests

FCMAB121F

Milli-Mark: Cell Signaling Qty

Catalog No.

Mouse Anti-Akt/PKB-Alexa Fluor 647, clone SKB1

Human

Mouse

100 tests

FCMAB128A6

Phospho-Erk1/2 (Thr202/Tyr204, Thr185/Tyr187)-PE

Human

Rabbit

100 tests

FCMAB100P

Mouse Rat Anti-Ki-67-APC

Human

Mouse

100 tests

FCMAB103AP

Phospho-STAT1 (Y701) Alexa 488

Human

Mouse

100 tests

FCMAB106A4

Mouse Rat Anti-b-catenin-PE, clone 7F7.2

Mouse

Mouse

100 tests

FCMAB1209P

Phospho STAT5A/B (Y694/699) –PEH

Human

Mouse

100 tests

FCMAB105P

Mouse Bovine Sheep

25

Components of the Guava Flow Cytometry Solution Instruments

Traditional sheath fluid system Waste

Traditional

The guava easyCyte flow cytometry systems are uncomplicated instruments that deliver the power of

Sample Flow

Sheath Fluid: ~10 mL/test Waste: 8,000 mL per 8 hour run Typical #Cells Per Protocol: 100,000 1,000,000 cells/test

multiplexed cell analysis—right on your benchtop. The culmination of over a decade of flow cytometry expertise, these instruments use minimal sample, generate less

Laser

waste, and are easier to use and maintain than traditional flow cytometers—all while providing the power you need

Waste

in the most compact format available. These advantages

Laser

are made possible by our patented microcapillary flow cell

Sample

Injection Tip

Sheath Fluid

Sheath Flow

technology.

Microcapillary Flow Cytometry Technology At the heart of every guava easyCyte system is a unique, microcapillary flow cell that eliminates the need for sheath fluid. This translates into smaller samples, less reagents

Guava-patented microcapillary system Sheath Fluid: None Waste: < 80 mL per 8 hour run Typical #Cells Per Protocol: 1,000 - 10,000 cells/test

and minimal waste—saving you both time and money.

Microcapillary

Waste

Because the flow cell is self-aligning and user-replaceable, you can remove it yourself at any time for cleaning and maintenance—no more expense or downtime for service visits. And by eliminating complicated microfluidics, we’ve created a compact to save valuable lab space and

Waste

eliminated much of the operational costs compared to Laser

sheath-based instruments.

Laser Sample Flow

Sample

Flow Cell

Sample

The patented Guava microcapillary allows for direct sampling,with no need for sheath fluid and complicated microfluidics. The result is a compact, easy to use system whose operational costs are a fraction of a sheath-based instrument.

26

guava easyCyte High Thoughput Sampling Instruments

Microcapillary flow cell requires no sheath fluid and is user-replaceable

Wash vial offers a high-pressure purge to easily clear obstructions from the flow cell

Up to six-color detection made possible by one (blue) or two excitation lasers (blue & red)

Waste vial collects less than 80 mL of waste in a typical 8-hour workday

Small footprint saves valuable laboratory space Width: 20.3 in (51.5 cm)

Robotic sample tray provides walk-away automation for a 96-well microplate and up to 10 sample tubes

Depth: 23.4 in (59 cm) Height: 10.0 in (25.4 cm) (does not include laptop)

Specifications Specifications

System Catalogue # Option # Laser Laser Wattage

easyCyte 5HT

easyCyte 6HT

easyCyte 6HT-2L

easyCyte 8HT

0500-4005

0500-4005

0500-4007

0500-4008

N/A

0500-4006

N/A

N/A

Blue (488 nm)

Blue (488 nm)

Blue (488 nm) and Red (640 nm)

Blue (488 nm) and Red (640 nm)

20 mW

20 mW

40 mW

75 mW

FSC SSC Green

525/30 nm

525/30 nm

525/30 nm

525/30 nm

Yellow

583/26 nm

583/26 nm

583/26 nm

583/26 nm

Red1

680/30 nm

680/30 nm

690/50 nm

690/50 nm

NIR1

N/A

785/70 nm

N/A

785/70 nm

Red2

N/A

N/A

661/19 nm

661/19 nm

NIR2

N/A

N/A

N/A

785/70 nm

Microcapillary Fluidics Direct, Absolute Cell Counts Automation – 96 Well and 10 Tubes Mixing Dell® Latitude® E6520 Laptop with Intel® Core InCyte™ Software guavaSuite Software Modules Digital Signal Processing

27

guava easyCyte Single Sample Instruments

Microcapillary flow cell requires no sheath fluid and is user-replaceable

Single sample loader Swivel arm functionality, holds two tubes and allows instant acquisition

Up to six-color detection made possible by one (blue) or two excitation lasers (blue and red)

Waste vial collects less than 80 mL of waste in a typical 8-hour workday

Small footprint saves valuable laboratory space Width: 17.75 in (45.1 cm)

Wash vial offers a high-pressure purge to easily clear obstructions from the flow cell

Depth: 17.25 in (44.5 cm) Height: 8.75 in (22.2 cm) (does not include laptop)

Specifications Specifications

System

easyCyte 5

easyCyte 6

easyCyte 6-2L

easyCyte 8

Catalogue #

0500-5005

0500-5005

0500-5007

0500-5008

N/A

0500-5006

N/A

N/A

Blue (488 nm)

Blue (488 nm)

Blue (488 nm) and Red (640 nm)

Blue (488 nm) and Red (640 nm)

20 mW

20 mW

40 mW

75 mW

525/30 nm

525/30 nm

525/30 nm

525/30 nm

Option # Laser Laser Wattage FSC SSC Green Yellow

583/26 nm

583/26 nm

583/26 nm

583/26 nm

Red1

680/30 nm

680/30 nm

690/50 nm

690/50 nm

NIR1

N/A

785/70 nm

N/A

785/70 nm

Red2

N/A

N/A

661/19 nm

661/19 nm

NIR2

N/A

N/A

N/A

785/70 nm

Automation – 96 Well and 10 Tubes

N/A

N/A

N/A

N/A

Mixing

N/A

N/A

N/A

N/A

Microcapillary Fluidics Direct, Absolute Cell Counts

Dell Latitude E6520 Laptop with Intel Core InCyte Software guavaSuite Software Modules Digital Signal Processing

28

Software Your specific research needs are always changing and EMD Millipore’s guava software is uniquely adaptable to accommodate you at every level. The guavaSoft application-specific modules have plug-and-play formats designed for our optimized reagents. For more flexible, user-defined formats, InCyte software delivers many highlevel features which uniquely enable easy visualization of data in a broad biological context. All our software modules use the same intuitive user interface, to make it easy to switch from one format to another. You can export data quickly to spreadsheets or any thirdparty analysis format. Moreover, our software packages can enable 21 CFR part 11 compliance.

29

InCyte: Intuitive InCyte software brings a new level of analytical power to

a single sample. One of its uniquely powerful benefits is the

flow cytometry. It is the first solution designed to empower

ability to display comparative results and the experiment

every user to draw conclusions about the biological

level, using features like heat maps and IC50/EC50 curves

significance of data.

which allow easy target identification. Automated compensation reduces the amount of time to analyze

Its intuitive, easy-to-use interface makes it possible to

complex multicolor assays by providing an automatic

visualize and compare up to eight data sets at once, with

correction of the spectral overlap of simultaneously

drag and drop features to simplify the set up of gating

analyzed dyes. As a result, InCyte software can function as

strategies. Many high-level analysis features are already

the primary data acquisition and analysis package for the

built in. Entire experiments can now be analyzed and

instrument.

viewed at once, in less time than it usually takes to analyze

Drag-and-drop gating allows selection of specific populations for further analysis, with the ability to highlight groups using color back-gating

IC50 curves show inhibition range of a dose-response curve

View up to 11 plots all at once, with real-time plot adjustments

Organize acquired data in this panel

G

F

H

Easily create analysis templates

D E

Quickly link to and review previously analyzed data

Heat mapping allows rapid visualization of up to 6 parameters at once, within a single plate or across multiple experiments

30

Construct heat maps or EC50/ IC50 curves by selecting groups of data and using slider bars to set cut-offs or threshold values

product HIGHLIGHTS

Scepter™ 2.0 The next generation of automated cell counting Scepter, the first handheld automated cell counter, brought portable and precise cell counting to the palm of your hand Scepter 2.0 now expands your ability to precisely count particles as small as 3 μm in diameter for compatibility with additional cell types such as: stem cells, blood cells, and yeast. Learn More: www.millipore.com/scepter

Versatility in a Complete Package. MILLIPLEX® map magnetic bead-based immunoassay kits & MAGPIX®® instruments Based on the Luminex® xMAP® technology and EMD Millipore’s 25 years of experience, we provide multiplex kits and ELISAs in key research areas, enabling you to perform multiple assays in a single sample. Learn more of our solutions for Cancer, Cellular Metabolism, Neuroscience, and Toxicity at: www.millipore.com/magbeads

31

To Place an Order or Receive Technical Assistance In the U.S. and Canada, call toll-free 1-800-645-5476 For other countries across Europe and the world, please visit www.millipore.com/offices For Technical Service, please visit www.millipore.com/techservice

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www.emdmillipore.com

EMD Millipore, the M logo, Cell Cycle Stop, ChemiScreen, easyCyte, InCyte, and Scepter are trademarks of Merck KGaA, Darmstadt, Germany. guava, ViaCount, and Milli-Mark are registered trademarks of Merck KGaA, Darmstadt, Germany. All trademarks belonging to third parties are the property of their respective owners. Lit No. PB3322EN00 BS-GEN-11-04588 Printed in the USA 03/2013 © 2013 EMD Millipore Corporation, Billerica, MA USA. All rights reserved.