T cell preparation by rosetting FGB

Revised 7/31/96

T cell preparation by rosetting

General information: all spins are at 4 °C with brake full ON (except Ficoll which is at RT with brake full OFF). RPMI is RPMI 1640 4+ (500 mL RPMI 1640, 50 mL HI FBS, 5 mL Pen Strep, 5 mL L-Glutamine, 560 µL 2-ME working solution [8.3 mL HBSS / 25 µL 2-ME stock])

Note: see the protocol titled "Isolation of peripheral blood cells" to see how to obtain a sample of T cells from fresh blood as they are to be used here.

Reagents: 50 mL, 40% blood / 60% Alsever's solution from American Red Cross, Durham, NC. SRBC's from freezer in 491. Cold HBSS. RPMI 4+ medium. FICOLL.

1. Place 25ml (or 50ml) of sheep red blood cells (SRBC) then 20ml of cold HBSS into a 50 mL tube per tube to be prepared and spin at 2000. rpm for 10 minutes at 4 °C.

2. Wash 2X with 20 ml 1X PBS or 1X HI-FBS (4 °C) (i.e. to pellet add 20 mL PBS or HI-FBS, mix well and spin at 2000 rpm for 10 minutes.

3. Suck off PBS or HI-FBS leaving the pellet which should occupy 5ml (or 10ml, see above).

4. Resuspend in HI-FBS to make 25% SRBC (i.e. for a 10 ml pellet add 40 ml HI-FBS; thus 5ml would = 20ml).

Non-Adherent (NA) peripheral blood cells.

Continued from step 22 in the isolation of PCB's.

1. Spin down cells at 1000 rpm for 10 minutes at 4 °C (or 1200 rpm for 8 minutes) then suck off supernatant.

2. Resuspend cells in 20-30 ml RPMI medium, mix and pool if >1 tube.

3. Count cells and make up to 20 x 10 6 cells per ml. use hemacytometer if necessary.

4. Add 20 ml NA cells to 5 mL 25% SRBC.

Note: Do not disturb rosette steps 5-9.

5. Spin at 700 rpm for 10 minutes at 4 °C to allow rosette formation.

6. Mix by inversion and spin at 1000 rpm for 10 minutes at 4 °C.

7. Keep on ice for 20 minutes.

8. Suck off medium leaving around 7 mL and gently bring up to 30ml with RPMI, mix gently.

9. Ficoll cells (30 ml cells + 17 ml Ficoll). Ficoll is at RT in 491. Apply Ficoll gently, from bottom of tube up, so as not to disturb interphase.

10. Spin at 2000 rpm for 25 minutes at RT with brake OFF.

11. Aspirate supernatant, interphase and ficoll, collect the pellet on the bottom (the loose pellet of SRBC and T cells) and resuspend in RPMI to make up to 25-30 ml.

12. Spin down at 1000 rpm for 10 minutes at 4 °C.

13. Aspirate supernatant then loosen pellet by tapping on table.

14. Gently, yet quickly resuspend cells in 10-20 ml cold 0.2% NaCl (hypotonic solution, isotonic is 0.9% NaCl). Do this step in less than 30 seconds mixing well.

15. Add 10-20 ml 1.6% NaCl (hypertonic solution).

16. Mix red suspension with pipet and immediately spin at 1000 rpm/10 minutes at 4°C.

17. Aspirate supernatant then resuspend white pellet with a pipet in 20 ml RPMI.

18. Spin at 1000 rpm for 10 minutes.

19. Repeat steps 17 and 18 two more times.

20. Count cells; see below.

21. Resuspend cells in 20 mL RPMI (Generally 2-4x106 cells/mL for culture).

22. Add 40 mL RPMI to 2 tissue culture (TC) flasks.

23. Add 10 mL cells to each flask.

24. Label and incubate overnight at 37 °C.

- dead cells take up dye, not live cells.

a. To an eppendorf tube, add 100 µL Trypon Blue and 25 µL of cells.

b. Put on hemocytometer (drawer in TC room).

c. Count 4 corners and middle. Be consistent in counting bordering cells.

d. Cell count per mL = (#cells in 4 corners plus middle) x 5 x 51 x 1000 (this is essentially finding the number of cells in all 25 squares and multiplying by 51000).