Biology Reference
In-Depth Information
6. Centrifuge the cells at 500 ×
g
for 10 min.
7. Resuspend the cell pellet in PBS and wash it two more times to
assure removing the remaining peptide.
8. Resuspend the cells in PBS at a concentration of 5 × 10
7
cells/
mL.
9. Thaw an aliquot of 5 mM stock CFSE solution.
10. Make a fresh CFSE
low
stock solution by diluting 5 mM stock
1/10 in DMSO.
11. Incubate the unpulsed target splenocytes with the higher con-
centration of CFSE (CFSE
high
): add 1
L of the 5 mM stock
CFSE for each mL of peptide-pulsed target splenocytes (fi nal
concentration of 5
μ
μ
M). With a pipette resuspend cells
homogeneously.
12. Incubate the peptide-pulsed splenocytes with the lower con-
centration of CFSE (CFSE
low
): add 1
L of 0.5 mM stock
CFSE for each milliliter of control-pulsed cells (fi nal concen-
tration of 5
μ
M). Mix with a pipette.
13. Incubate the cells at 37 °C for 10 min. Periodically agitate the
cells.
14. Add 10 volumes of pre-warmed RPMI to the CFSE-labeled
cells to stop the reaction.
15. Centrifuge cells at 500 ×
g
for 5-10 min at 4 °C.
16. Remove the supernatant and resuspend the pellet in RPMI.
Wash two more times with RPMI.
17. Count the mononuclear cells by trypan blue exclusion using a
hemocytometer.
18. Resuspend the cells in PBS at a concentration of 5 × 10
7
mononuclear cells per milliliter.
19. Combine an equal volume of peptide-pulsed CFSE
low
cells
with control-pulsed CFSE
high
cells.
20. Inject intravenously 200
μ
L of the combined cell populations
in to the tail vein of each recipient animal.
21. After 20 h harvest spleen from the recipient mouse injected
with CFSE-labeled target cells following
steps 1-10
of
Subheading
3.9
.
22. Resuspend the cells in FACS buffer.
μ
23. Proceed to fl ow cytometry to determine the percentage of cells
having low and high content of CFSE. CFSE can be analyzed
using a fl ow cytometer equipped with 488 nm excitation and
emission fi lters. Set the fl ow cytometry gates using a naïve
recipient mouse (not treated with adenovirus) than has been
injected with the two target populations.
