Biology Reference
In-Depth Information
28. Add 200
L/well of Perm Wash. Spin the plate at 400 ×
g
4 °C
for 5 min. Remove the SN. Vortex. Repeat this wash.
29. Resuspend cells in 100
μ
L/well of FACS Fixative, transfer
samples to FACS tubes, fi ll up to 300
μ
L with FACS Fixative.
30. Keep at 4 °C, dark, and measure fl uorescence staining by FACS
within 48 h.
μ
3.10 CTL Responses
by ELISPOT
1. Wash an ELIspot 96-well plate fi ve times with 200
μ
L/well of
sterile water (
see
Note 8
).
2. Add 100
L/well of coating antibody solution and incubate
overnight at 4 °C.
3. Isolate splenocytes following
steps 1
-
10
of Subheading
3.9
.
4. Remove coating antibody and wash with 200
μ
μ
L/well of PBS.
5. Block membranes with 200
L/well of RPMI/10 % FBS.
Incubate at least for 30 min at RT.
6. Dilute splenocytes at 2.5 × 10
6
/mL. Add 100
μ
μ
L of cells/well.
7. Add 100
L/well): (a)
PMA + Ionomycin as positive control. Final concentration:
15 ng/mL PMA + 250 ng/mL Ionomycin; (b) test peptides
(CTL epitopes) at 2
μ
L of stimulants (fi nal volume 200
μ
M fi nal concentration; and (c) RPMI
medium (or irrelevant peptide) as negative control.
8. Incubate at 37 °C 5 % CO
2
at least 16 h. Do not move the
plate during this time.
9. Remove the cells and wash fi ve times with 200
μ
μ
L/well of PBS.
10. Add 100
μ
L/well of detection antibody solution and incubate
2 h at RT.
11. Wash fi ve times with 200
μ
L/well of PBS.
12. Add 100
μ
L/well of Streptavidin-ALP solution and incubate
1 h at RT.
13. Wash fi ve times with 200
μ
L/well of PBS.
14. Add 50
L/well of BCIP/NBT solution and develop until dis-
tinct spots emerge (10-30 min).
15. Stop color development by washing with tap water.
16. Leave the plate to dry. Read spots with an ELIspot plate reader.
μ
3.11 In Vivo
Cytotoxicity Assay
1. Isolate splenocytes following
steps 1-10
of Subheading
3.9
.
2. Divide cells into two tubes: “peptide-pulsed” and “non-pulsed
cells”.
3. To the pulsed cells add peptide at fi nal concentration of
1-2
M. Add PBS an equivalent volume to the non-pulsed
target cells.
4. Incubate the cells in a 37 °C water bath for 1 h.
5. Wash the cells with RPMI medium.
μ
