Reagents:

• HIV-1 LAI viral stock (CEM cells infected at MOI of 0.01, day 3 fresh media, day 4 harvest supernatant, store at –70°C: 10⁷ TCID₅₀/ml).

• Mock stock (supernatant from uninfected CEM cells treated as above).

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

• 50 ml conical tubes.

• Hemocytometer.

• Trypan Blue.

• Sterile PBS.

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

• 18-gauge needle and 20 ml syringe

Protocol:

A.Infection:

1. Expand T cell line 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 LAI and 25 x 10⁶ for mock), 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 cells at 10 x 10⁶ cells/ml cRPMI in a 50ml tubes (e.g. 25 x 10⁶ cells in 2.5 mls).

10. For each time point, add LAI stock at multiplicity of infection (MOI) of 1 (250 µl stock = 25 x 10⁶ TCID₅₀ for 25 x 10⁶ cells) to one aliquot and same volume of mock to the other aliquot.

11. Incubate at 37°C for 1 hour in shaking waterbath.

12. Add sterile PBS to 50 ml.

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

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

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

16. Resuspend pellet in cRPMI at 1 x 10⁶ cells/ml in T-175 flask.

17. Incubate at 37°C, 5% CO₂.

B. Harvest:

18. Harvest cells from both LAI- and mock-infected cultures at the appropriate time points, e.g. 24 hours, 48 hours. Transfer each cell suspension to separate 50 ml tubes.

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

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

21. Take sample of cell suspension for counting (as above). Count % dead cells.

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

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

24. Resuspend each cell pellet at a concentration of 1 x 10⁶ cells/ml PBS.

25. Take two 500 µl aliquots for analysis of p24 expression by FACS (see appropriate protocol).

26. Then add PBS up to 50ml to the rest of the cells.

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

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

29. Add appropriate amount of solution D (with β-ME) to each cell pellet (see total RNA isolation protocol).

30. Vortex until entire pellet is resuspended.

31. Pass mixture 5 times through 18-gauge needle with 20ml syringe.

32. Isolate RNA or store at -70°C.

     

    Notes:

    * 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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