Reagents:

• sterile TC PBS.

• Tat protein dissolved in sterile TC PBS (25 µg/ml).

• BSA dissolved in sterile TC PBS, filtered over 0.22 µm filter (25 µg/ml).

• cRPMI (10% FBS, L-gluatmine, Penicillin/Streptomycin)

• Hemacytometer.

• Trypan Blue.

• Solution D: use 200 µl per 10⁶ cells (add 7.2 µl /ml RNAse-free β-Mercaptoethanol just before use).

Protocol:

1. Expand Jurkat cells until the appropriate number of cells has been obtained (3-4 days after passage the cell suspension will be approximately at 10⁶ cells/ml).

2. Need 25 x 10⁶ cells per condition to get sufficient RNA. So for one time point need 50 x 10⁶ cells [25 x 10⁶ for Tat and 25 x 10⁶ for control (=BSA)], two time points 100 x 10⁶ cells, etc.

3. Spin down cells at 1200 rpm for 5 minutes at RT.

4. Resuspend cell pellet in 10 ml cRPMI.

5. Take sample of cell suspension for counting.

6. Dilute sample to 0.5-1.0 x 10⁶ cells/ml and mix 1:1 with Trypan Blue.

7. Put 20 µl in hemacytometer and count immediately.

8. Calculate concentration of cell suspension^*.

9. For each condition, aliquot 25 x 10⁶ Jurkat cells in 25mls cRPMI in a T-75 flask.

10. For each time point, add 5 µg of Tat protein to one T-75 flask, 5 µg of BSA to the other flask.

11. Incubate at 37°C, 5% CO₂.

12. Harvest cells from both Tat- and control-flask at the appropriate time points, e.g. 24 hours, 48 hours. Transfer each cell suspension to separate 50 ml tubes.

13. Spin down cells at 1200 rpm for 5 minutes at RT.

14. Resuspend each cell pellet in 5ml PBS, and then add PBS up to 50ml.

15. Spin at 1200 rpm for 5 minutes at RT.

16. Discard supernatant. Remove last drops of remaining fluid with vacu-system.

17. Repeat steps 14-16.

18. Add 5 ml of solution D (with β-ME) to each cell pellet.

19. Vortex until entire pellet is resuspended.

20. Isolate RNA or store at -70°C.

     

     

    Note: 5 ml of solution D is added to each cell pellet assuming the cell numbers have doubled over 24 hours. You could also take a sample and count with the hemacytometer between the two PBS washes and adjust the volume of solution D used to the actual count.

    * If you count the 20 small squares in one block the calculation is as follows:

    Number of cells/ml = cell count x dilution factor x 10⁴

    Dilution factor includes both the dilution needed to get the cell suspension at approximately 10⁶ cells/ml and the 1:1 dilution with Trypan Blue (e.g. 10 fold dilution of cells, then Trypan Blue dilution gives a dilution factor of 20). If you count more than one block of 20 squares you need to divide the cell count by the number of blocks counted.

     

    For specifics on appropriate handling and waste procedures please see the online chemical SOPs or our waste and spill notebook located in room 352.

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