Biology Reference
In-Depth Information
6. Remove the supernatant and resuspend the cell pellet in PBS.
Wash the pellet two more times with PBS, discarding tissue
debris.
7. Resuspend the cells in 5 mL of RPMI medium.
8. Refi lter the cells through a cell strainer if clumps are present.
9. Count the mononuclear cells by trypan blue exclusion using a
hemocytometer.
10. Resuspend cells at 10
7
in RPMI.
11. Seed 100
L/well of the cell dilution (10
6
cells/well) in a
96-well plate with U-bottom.
12. Add 100
μ
L/well): (a) PMA
and Ionomycin as positive control. Final concentration: 30 ng/
mL PMA + 500 ng/mL Ionomycin; (b) Test peptides (CTL
epitopes) at 2
μ
L of stimulant (fi nal volume 200
μ
M fi nal concentration, and (c) RPMI medium
(or irrelevant peptide) as negative control.
13. Incubate cells at 37 °C 5 % CO
2
for 2 h to O/N.
14. Add Brefeldin A in the well to a fi nal concentration of 10
μ
g/
mL. Mix gently with the pipette. Incubate at 37 °C 5 % CO
2
for 4 h.
15. Spin the plate at 400 ×
g
4 °C for 5 min. Check visually if there
is pellet.
16. Remove the SN by fl icking the plates and then blotting once
on clean paper. Check visually if there is pellet.
17. Vortex (check that pellet is well resuspended).
18. Add 50
μ
μ
L/well of antibody mix. Resuspend cells gently by
pipetting.
19. Incubate 30 min on ice, dark (cover the plates with aluminum
foil). Alternatively cells can be incubated O/N in the fridge.
20. Add 150
L/well of FACS Buffer. Spin the plate at 400 ×
g
4 °C for 5 min. Remove the SN. Vortex. Repeat this washing
step two additional times.
21. Permeabilize cells adding 100
μ
L/well of 2 % formaldehyde in
cold PBS. Resuspend cells gently by pipetting.
22. Incubate 20 min on ice.
23. Add 100
μ
L/well of Perm Wash. Spin the plate at 400 ×
g
4 °C
for 5 min. Remove the SN. Vortex.
24. Add 200
μ
L/well of Perm Wash. Spin the plate at 400 ×
g
4 °C
for 5 min. Remove the SN. Vortex. Repeat this washing step.
25. Add 50
μ
L/well of second antibody mix (prepared in Perm
Wash). Resuspend cells gently by pipetting.
26. Incubate 30 min on ice, dark.
27. Add 150
μ
L/well of Perm Wash. Spin the plate at 400 ×
g
4 °C
for 5 min. Remove the SN. Vortex.
μ
