TBARs Protocol

TBARs Protocol:

Reagents     

Trypsin Completed Media GTP Lysis Buffer with Inhibitors 0.67% TBA in 20% Trichloroacetic Acid       Must be made fresh each time MDA Stock for Standards       Light sensitive Protein Assay Reagents

Protocol1. Trypsinize cells to detach them from the plates (2 (2 mL of trypsin for 6 cm dishes). a. Neutralize Neutrali ze with complete media (2 mL of of media for 6 cm dishes). b. Spin down down at 1000g for 5 minutes. c. Aspirate trypsin-containing trypsin-con taining media. 2. Before spinning down the cells, remove 10 QL from each plate to count cell numbers using the Trypan Blue method with a hemacytometer (this is a good step to do while the cells are being spun down). a. Add 10 QL of the trypsinized cells to 10 QL of Trypan Blue (in 1.5 mL tubes in the drawer with the hemacytometers). b. Add 10 QL of the new mixture to a hemacytometer and determine the number of  cells (use the hemacytometer protocol if you are unfamiliar with the method). 3. Calculate Calcula te which which plate had the fewest number of cells, and then make dilutions using GTP lysis buffer to ensure all treatments will have an equal number of cells for the same volume. a. 250 QL is the minimum amount of GTP lysis buffer that should be added to the pellet of the treatment with the fewest cells. b. At this point, you may also want want to turn on the heating block in Dr. Cremo¶s Cremo¶s laboratory (use the ³high´ setting). 4. Using a 26.5 gauge gauge syringe, shear the cell pellet in GTP lysis buffer by passing through the needle 10 times. a. Try and minimize the creation of bubbles to conserve volume. 5. Add 125 QL of this lysate to a new 1.5 mL tube. a. MAKE SURE TO TO ALSO HAVE AT LEAST ONE TUBE WITH 125 QL OF LYSIS BUFFER TO USE AS A BLANK FOR THE ASSAY. b. You may also want to use MDA standards if you you are interested in knowing the actual level of MDA instead of just relative percentages. 6. Add 125 QL of freshly made 0.67% TBA in 20% TCA to the 125 QL of sample. 7. Place the tubes on the heating block (you want it at least 100ºC) for 20 minutes. a. Put the safety safety caps on the the tubes to prevent them from popping open during this this step.

8.

Place the tubes on ice for 10 minutes. 9. Spin down the samples in the cold room centrifuge at top speed (13) for 5 minutes. 10. Add 200 QL of supernatant from each tube to a 96-well plate. a. You want to use the same type of plate you would use for a protein assay, not a culture plate. 11. Read the 96-well plate at 53 8 nM in Dr. Schooley or Dr. Cremo¶s laboratory. a. This value is your absolute reading and indicates the amount of MDA formed. 12. Perform data analysis and create graphical representations.

*Alternatively, cells can be scraped with GTP lysis buffer using the traditional protocol, but DO NOT spin down the sample (most of the lipids are in what would pellet out). These samples should be diluted after doing a protein assay to standardize their input.