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Protocol
Human Lymphocyte Culture and Chromosome Analysis Peter Benn1 and Judith Delach Human Genetics Laboratories, Department of Genetics and Developmental Biology, and Department of Pathology and Laboratory Medicine, University of Connecticut Health Center, Farmington, CT 06030-6140, USA
INTRODUCTION Phytohaemagglutinin (PHA), a lectin derived from the red kidney bean, is a powerful mitogen for human T-cells. When PHA is added in vitro to whole blood, mitotic cells can be found after 48 h, with a peak mitotic index at ~64-72 h. The convenience of peripheral blood as a source of human cells, the abundance of mitotic cells, and the simplicity of the cell culture technique make this the most convenient approach to study human chromosomes for both clinical and research purposes. This method of chromosome preparation provides metaphase cells that can be stained by a variety of methods or used for fluoresce fluorescence nce in situ hybridiz hybridization ation (FISH). The most common chromoso chromosome me staining techniqu techniques es involve exposing fixed preparations to a protease (e.g., trypsin), followed by an appropriate semipermanent stain. The characte characteristic ristic banding patterns obtained reflect both structu structural ral and functiona functionall differences in different parts of the chromosomes. The staining procedure described here provides a Giemsa banding pattern pattern using trypsin with Wright stain (i.e., GTW banding). This procedure procedure is reliable and, with only minor modifications, suitable for preparing chromosomes from a variety of human tissues.
RELATED INFORMA INFORMATION TION Methods to optimize lymphocyte culture for suboptimal specimens and additional tips for cell culture and slide preparation are provided by Clouston (2001) and in the AGT Cytogenetics Manual (see Barch Barc h et al. 1997). Alternative Alternative staining staining tech technique niquess that can be used to iden identify tify specific chromosome chromosome regions are reviewed by Benn and Tantravahi (2001). Many of these alternative staining methods are now only rarely used because FISH with specific probes can be more informative (see the article at http://www.utoronto.ca/cancyt http://www .utoronto.ca/cancyto/protocols_software/cyt o/protocols_software/cytogenetics/FISHpdf. ogenetics/FISHpdf.pdf). pdf). For additional examples of human chromosome banding patterns and a description of the nomenclature that should be used use d to des descr cribe ibe hum human an ka karyo ryoty type pes, s, se see e th the e Int Inter ernat nation ional al Sy Syst stem em for Hum Human an Cy Cytog togene enetic tic Nomenclature (Shaffer and Tommerup 2005).
MATERIALS CAUTIONS AND RECIPES: Please see Appendices for appropriate handling of materials marked with , and
recipes for reagents marked with .
Reagents Blood (5 mL) Collect and store blood in green-top (i.e., sodium heparin-containing) Vacutainer tubes. Carnoy’s fixative (freshly prepared)
Colcemid (KaryoMAX, 10 µg/mL; Invitrogen 15212-012)
1
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[email protected]) Please cite as: CSH Protocols; 2008; doi:10.1101/pdb.prot5035 © 2008 Cold Spring Harbor Laboratory Press
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Ethidium bromide (1 µg/mL; Sigma-Aldrich) Fetal bovine serum Gurr’s buffer solution Dissolve one Gurr pH 6.8 buffer tablet in 1 L of H 2O. Store at 4°C.
Hank’s balanced salt solution (10X, calcium- and magnesium-free) Hypotonic solution (prewarmed to 37°C) Mounting medium (e.g., Pro-Texx or comparable) Phytohaemagglutinin (PHA) (reagent grade; Remel R30852701) RPMI (complete) Other complete tissue culture media (e.g., Ham’s F10, Eagle’s Minimal Essential Medium, etc.) are also suitable. Trypsin solution (5%, w/v; Difco Bacto 250, #215240, Becton Dickinson) Wright stain
Equipment Beaker containing hot H2O Centrifuge (benchtop, 200g 200g ) Coverslips Gloves (latex) Hair dryer Hood (tissue culture, laminar flow) Incubator preset to 37°C, 5% CO 2, 100% humidity Karyotyping station (automated; e.g., Applied Imaging Cytovision, Metasystems, etc.) Microscope (phase contrast, low power) Oil (immersion) Photomicroscope equipped with 10X and 100X objectives A high quality microscope is essential for effective chromosome analysis.
Pipettes (Pasteur, disposable) Pipettes (sterile, graduated, disposable, 5-mL) Slide support (horizontal) (e.g., two glass rods or pipettes, ~2 in. apart) Slide warmer, hotplate, or oven preset to 56°C-60°C Slides (microscope) Wash in H 2O and store at 4°C.
Staining jars (Coplin) Tubes (polypropylene, sterile, 10-mL) Vortex mixer (optional; see Step 10) Water bath preset to 37°C
METHOD Lymphocyte Culture 1. Working under the tissue culture hood and using sterile technique, aliquot complete RPMI into
three polypropylene tubes, 4.5 mL per tube. With multiple cultures, harvesting can be carried out at different times and cultures with the greatest number of high quality metaphases can be used for analysis. 2. Add 0.5, 0.45, or 0.4 mL of whole blood to each tube. tube. 3. Add 0.1 mL of PHA to each tube. 4. Tighten the caps. Mix by inverting several times. Loosen the caps sufficiently to allow for exchange
of gases. 5. Place in the incubator incubator for 64-72 h at 37°C.
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6. Two hours before harvesting, add 50 µL of ethidium bromide to each tube. Return the tubes to
the incubator. Ethidium bromide inhibits chromosome contraction, facilitating the analysis of longer chromosomes with higher band resolution. Depending on the objective of the analysis, ethidium bromide may not be needed. 7. Forty-five minutes before harvesting, add 50 µL of colcemid solution to each tube. Return the
tubes to the incubator. Cell Harvesting 8. Remove the tubes from the incubator. incubator. Centrifuge at 200g 200g for for 8 min. 9. Carefully remove as much of the supernatant as possible with a Pasteur pipette, taking care not to
disturb the white cell layer (i.e., buffy coat) above the red cell pellet. 10. Mix the red and white cell layers with a Pasteur pipette or vortex mixer. 11. Slowly add 1 mL of hypot hypotonic onic solution solution (prewarmed (prewarmed to 37°C) dropwise, dropwise, mixing well with the cell
suspension. Continue to add hypotonic solution to a final total volume of 10 mL. As the volume in the tube increases, the rate at which the hypotonic solution is added can be increased gradually. 12. Incubate in a water bath at 37°C for 10 min. 13. Add 1 mL of freshly prepared Carnoy’s fixative to each tube. Cap the tubes. Mix by inverting.
200g for for 8 min. Carefully remove the supernatant with a Pasteur pipette, taking care 14. Centrifuge at 200g not to disturb the cell pellet. 15. Break up the pellet by agitation. Add 0.5 mL of Carnoy’s Carnoy’s fixative, dropwise, mixing after each drop.
Continue to add fixative to a final total volume of 5 mL. As the volume in the tube increases, the rate at which the fixative is added can be increased gradually. 16. Repea Repeatt Steps 14 and 15 twice more. Resuspend Resuspend the final pellet by dropw dropwise ise addition addition of Carnoy’s Carnoy’s
fixative to a final total volume of ~1 mL. If necessary, preparations can be stored in a tightly capped tube with excess fixative at -20°C until slide preparation.
Slide Preparation 17. Remove a slide from the refrigerator. refrigerator. Label as appropriate. 18. Caref Carefully ully allow six to nine drops of the cell suspension suspension from Step 16 to fall onto the slide. 19. Hold the slide over a beaker of hot H2O for 3-5 sec. The H 2O in the beaker should be just hot enough to produce a visible film of vapor on the slide. 20. Place the slide slide on the slide warmer warmer for ~30 sec to dry dry.. 21. Examine the slide under a phase contrast microscope to evaluate the quality of the preparation
and the extent of chromosome spreading. If the quality is satisfactory, prepare additional slides using the same technique. See Troub Troubleshooting. leshooting. 22. Incubate the slides at least overnight (or up to 3 d) on a 56°C-60°C slide warmer, hot plate or
oven. Omit this step if the slides are to be used for FISH.
GTW Bandin Banding g 23. Prepare the staining jars: i. Arran Arrange ge four Coplin Coplin staining staining jars in a line. ii. In each jar, dilute dilute 5 mL of 10X Hank’s Hank’s balanced salt solution solution with 45 mL of ster sterile ile H2O. iii. To the second jar, add 1 mL of the 5% trypsin solution. (This is the working trypsin
solution.)
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jar, add 2 mL of fetal bovine serum. (This (This is the arresting arresting solution.) solution.) iv.. To the third jar, iv 24. Place the horizontal horizontal slide support across across an open sink such that slides can be suppo supported rted but also
easily rinsed. 25. Rinse the slide slide to be stain stained ed in the first jar (first rinse) for 1-2 min. 26. Tran Transfer sfer the slide to the second jar (working trypsin solution) for ~30 sec. The amount of time needed for trypsin digestion can vary. 27. Place the slide in the third jar (arre (arresting sting solution) solution) for ~1 min. 28. Rinse the slide in the fourth jar (second rinse) for ~1-2 min. 29. While the slide is the fourth jar, prepare a working stain solution by mixing 0.5 mL Wright stain
with 2 mL of Gurr’s buffer per slide. 30. Tran Transfer sfer the slide from the fourth jar to the horizontal slide support. Gently flood the slide with the
stain.. Stain for ~40-5 stain ~40-50 0 sec. The amount of time needed for staining can vary. 31. Rinse off the stain with H2O. Dry the slide completely using a hair dryer. 32. Place two drops of mount mounting ing medium on the slide slide.. Carefully add a cover coverslip, slip, ensuring ensuring that no air
bubbles are present. See Troub Troubleshooting. leshooting. 33. Allow the coverslip to dry for 2-3 min before examining the sample under the microscope.
Chromosome Analysis 34. Identify individual well-spread and banded cells at low magnification (e.g., using a 10X objective,
equivalent to ~125X total magnification). See Troub Troubleshooting. leshooting. 35. Analyze suitable preparations at high magnification (e.g., (e.g., 100X objective with oil emersion, equiv-
alent to ~1250X total magnification). 36. For routine diagnostic diagnostic cytogenetic cytogenetic analysis, analysis, examine at least 20 cells to help rule out mosai mosaicism. cism. A much greater number of cells must be scored for chromosome breakage studies. 37. Using an autom automated ated karyotyping karyotyping stati station, on, digit digitally ally recor record d highhigh-resol resolution ution images of indiv individual idual
metaphase cells (Fig. 1). Sort and arrange the chromosomes into a karyotype (Fig. 2). Alternatively, cells can be photographed, printed, and each chromosome cut from the picture and arranged into a karyotype. Recognition of normal and abnormal chromosomes requires considerable skill. The assistance of an experienced cytogeneticist is recommended.
FIGURE 1. Lymphocyte metaphase cell with ~550 bands per haploid set of chromosomes.
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FIGURE 2. Karyotype prepared from the metaphase cell shown in Fig. 1.
TROUBLESHOOTING Problem: There is a large amount of debris on the slide, or too few, or too many, cells on the slide. [Step 21] Solution: To reduce debris, add additional fixative to the cell suspension, centrifuge, remove the super-
natant and resuspend the cells in fresh fixative. Adjust the final amount of fixative to obtain an appropriate cell density on the slide. Problem: There are no metaphase cells and few blast cells. [Step 21] Solution: This is caused by cell culture failure or if cells did not respond to the PHA. Some clinical
conditions are associated with a poor response to PHA. Ensure the following: 1. The cell culture medium medium and PHA are fresh. 2. The blood sample was collected in the correct sodium heparin anticoagulant tube. 3. The temperature and environment in the incubator are correct. 4. The tubes used for the incubation are nontoxic. Problem: Chromosomes from metaphase cells remain trapped in the cytoplasm; cells have a halo of
cytoplasm when viewed under the phase contrast microscope; the chromosomes are not sufficiently spread apart; or the chromosomes are too far apart, with many incomplete metaphase cells. [Step 21] Solution: Numerous factors affect the spreading of the chromosomes. These include the hypotonic
treatment, cell bursting if the hypotonic solution or fixative are added too rapidly, the force with which the cell suspension hits the slide, residual water in the cell or on the slide (which, in turn, is dependent on humidity), and surface tension exerted on the cell as the fixative evaporates. Keep the following points in mind: 1. Insufficient spreading often can be corrected by the following: i. Drop the cell suspension suspension from a greater greater height onto the slide. slide. ii. Tilt the slide as the suspension suspension is dropped. iii. Change the rate of fixative evaporation, for example, by gently blowing on the slide as it
dries. 2. Conversely Conversely,, broken metaphase cells with many missing chromosomes can be avoided by reducing
the above forces. 3. Use a hygrometer to monitor humidity. 4. If chromosomes remain trapped in cytoplasm even after the above adjustments, consider the
following: followi ng:
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sufficient hypotonic solution was used. i. Check whether sufficient ii. Check that hypotonic solution was prepared correctly. correctly. iii. Consider extending the hypotonic solution incubation time. 5. Developing a good preparation technique is largely a matter of trial-and-error, but once devel-
oped, should be reproducible. Problem: Metaphase chromosomes are too short, or very long with many overlaps that prevent analysis. [Step 21] Solution: Although more metaphase cells are seen in samples incubated in the presence of colcemid
for an extended time before harvesting, many of these will have chromosomes that are highly contracted and not optimal for analysis. Conversely, insufficient colcemid treatment results in fewer mitotic mitot ic cells with longer chrom chromosome osomess that are more difficult to analyz analyze e beca because use of overl overlaps. aps. Consider the following: 1. Change the time of incubation with colcemid. 2. If ethidium bromide was used, consider whether this is necessary, or if this incubation should also
be adjusted. Problem: Mounting medium is too viscous due to evaporation of the solvent. [Step 32] Solution: Dilute the mounting medium with Histo-Clear. Problem: Chromosomes are fuzzy or show regions with indistinct morphology. [Step 34] Solution: Consider the following: 1. Chromosome preparations may need to age longer before attempting banding. Extend the
incubation time at 56°C-60°C. However, aging the slides too long will make it difficult to obtain satisfactory banding. 2. Overexposure to the trypsin solution can overdigest the chromosomes. Overdigested chromo-
somes appear puffy, pale, and can have regions that are entirely missing. Problem: Chromosomes show few bands with little distinction between light and dark regions. [Step 34] Solution: Increase the duration of the incubation in the trypsin solution. Problem: Chromosome staining is too light or too dark. [Step 34] Solution: Increase or decrease the staining time, as necessary. The following will all affect the intensity
of staining: 1. Different batches of Wright stain 2. The time since the Wright stain was prepared 3. Exposure to light
DISCUSSION In addition to the analysis of human chromosomes derived from peripheral blood, diagnostic cytogenetic laboratories routinely make chromosome preparations from cultures of amniotic fluid cells, chorionic villus specimens, spontaneous abortion tissues, skin, bone marrow, and other human tissues. The cell culture methods used for these tissues are beyond the scope of this protocol. However, the methods used to make chromosome preparations are very similar and rely on harvesting the cells at times when there are optimal numbers of metaphase cells. For these tissues, the inclusion of ethidium bromide bromi de befor before e harves harvesting ting is usuall usuallyy omitte omitted. d. Table 1 lists suggested suggested durations for incub incubation ation in the presence of colcemid, hypotonic treatment, and trypsin treatment for these preparations. For research
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laboratories culturing human lymphocytes to evaluate the effects of potentially clastogenic agents, chromosome banding is usually unimportant. Ethidium bromide treatment and banding (i.e., Steps 6 and 22-28) can therefore be omitted. Finally, the lymphocyte cell culture technique described in this protocol is not suitable for rodent blood samples. Rodent lymphocytes do not respond to PHA; laboratories performing cytogenetic studies on these animals generally use bone marrow or short-term fibroblast cultures as a source of dividing cells.
Table 1. Recommended incubation times for chromosomal preparations Tissue
Colcemid solution (Step 7)
Incubation times Hypotonic solution (Step 12)
Trypsin solutiona (Step 26)
Cultured lymphocytes Amniotic fluid cells Chorionic villus cells Skin fibroblasts Spontaneous abortion tissues Bone marrow
45 25 25 25 25 30
10 45 45 45 45 15
~30 sec ~40 sec ~40 sec ~40 sec ~40 sec ~15-25 sec
min min min min min min
min min min min min minb
a
The amount of time needed for trypsin digestion can vary. See Troubleshooting. Hypotonic solution (for bone marrow cells) is designed specifically for use with bone marrow preparations and is recommended for this step. b
REFERENCES Barch, M.J., Knutsen, T., and Spurbeck, J.L., eds. 1997. The AGT cytogeneti gen etics cs lab labora oratory tory man manual ual , 3r 3rd d ed ed.. Li Lipp ppin inco cott tt-R -Rav aven en,, Philadelphia. Benn, P.A. P.A. and Tantr Tantravahi, avahi, U. 2001. Chromosome staining and banding techniques. In Human In Human cytogenetics: Constitutional analysis , 3rd ed. (ed. D.E. Rooney), Rooney), pp. 99–12 99–128. 8. Oxfor Oxford d Univ Universit ersityy Pres Press, s, Oxford.
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Clouston, H.J. 2001. Lymphocyte Clouston, Lymphocyte cult culture. ure. In Human cytogenetics: Constitutional analysis , 3rd ed. (ed (ed.. D.E D.E.. Roo Rooney ney), ), pp. 33–54. 33–54. Oxford University Press, Oxford. Shaffer Shaf fer,, L.G. and To Tomme mmerup, rup, N., eds. 2005. ISCN ISCN 200 2005: 5: An Internation Inter national al Syste System m for Human Cytog Cytogeneti enetics cs Nome Nomenclat nclature ure S. Karger AG, Basel, Switzerland.
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