6. Resuspend cells in approximately 25 ml of R10 (see Note 8).
Remove a small aliquot for cell counting and count the cells
using trypan blue or a similar cell counting protocol.
7. Add R10 to adjust the cell concentration to 10 Â 10
6 cells/ml.
Consider moving the cells to a T-75 flask instead of multiple
Falcon tubes to accommodate larger volumes; however, cells
would have to be transferred back to tubes for centrifuging.
8. Divide the cells into two equal portions in 50 ml Falcon tubes
or T-75 flasks. Label one “Target” and the other
““Nontarget.”
9. Prepare peptide solution: Make a working solution of peptide
of 2 mg/ml in DMSO (see Note 9).
10. Peptide pulse: To the “Target” cell sample tube only, add
OVA peptide (SIINFEKL) to a final concentration of 10 μg/
ml in R10 and gently agitate the solution to mix (see Note 10).
11. Incubate both “Target” and “Nontarget” cells for 30 min in a
water-jacketed incubator, 37
C, 5% CO 2 (see Note 11).
12. Centrifuge cells for 5 min at 400 Â g, 4
C in a swinging bucket
rotor to pellet cells, then discard supernatant.
In the following section all cells will be stained with a CFSE
pro-dye solution (high or low concentration) to a final cell concentration of 10 Â 10
6 cells/ml and a final CFSE pro-dye concentration of 2.5 or 0.25 μM.
13. Prepare CFSE pro-dye solutions at a 2Â concentration as
follows: (see Notes 2 and 12).
(a) CFSE
high solution: 5.0 μM CFSE in room temperature
(20–25
C) PBS.
(b) CFSE
low
solution: 0.5 μM CFSE in room
temperature PBS.
14. Separately resuspend “Target” and “Nontarget” cells in room
temperature PBS to a concentration of 20 Â 10
6 cells/ml.
Cover each tube/flask with tinfoil to prevent photobleaching
(see Note 13).
15. To the splenocytes, add an equal volume of CFSE
high or CFSElow as follows:
(a) CFSE
high solution is added to “Target” cells to obtain a
final conc. of 2.5 μM CFSE pro-dye.
(b) CFSE
low solution is added to “Nontarget” cells to obtain
a final conc. of 0.25 μM CFSE pro-dye.
16. Gently agitate the cells for 8 min at room temperature (see
Note 14).
17. Add an equal volume of ice-cold FBS to each tube and place on
ice for 5 min (see Note 15).
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Felicity C. Stark et al.
Remove a small aliquot for cell counting and count the cells
using trypan blue or a similar cell counting protocol.
7. Add R10 to adjust the cell concentration to 10 Â 10
6 cells/ml.
Consider moving the cells to a T-75 flask instead of multiple
Falcon tubes to accommodate larger volumes; however, cells
would have to be transferred back to tubes for centrifuging.
8. Divide the cells into two equal portions in 50 ml Falcon tubes
or T-75 flasks. Label one “Target” and the other
““Nontarget.”
9. Prepare peptide solution: Make a working solution of peptide
of 2 mg/ml in DMSO (see Note 9).
10. Peptide pulse: To the “Target” cell sample tube only, add
OVA peptide (SIINFEKL) to a final concentration of 10 μg/
ml in R10 and gently agitate the solution to mix (see Note 10).
11. Incubate both “Target” and “Nontarget” cells for 30 min in a
water-jacketed incubator, 37
C, 5% CO 2 (see Note 11).
12. Centrifuge cells for 5 min at 400 Â g, 4
C in a swinging bucket
rotor to pellet cells, then discard supernatant.
In the following section all cells will be stained with a CFSE
pro-dye solution (high or low concentration) to a final cell concentration of 10 Â 10
6 cells/ml and a final CFSE pro-dye concentration of 2.5 or 0.25 μM.
13. Prepare CFSE pro-dye solutions at a 2Â concentration as
follows: (see Notes 2 and 12).
(a) CFSE
high solution: 5.0 μM CFSE in room temperature
(20–25
C) PBS.
(b) CFSE
low
solution: 0.5 μM CFSE in room
temperature PBS.
14. Separately resuspend “Target” and “Nontarget” cells in room
temperature PBS to a concentration of 20 Â 10
6 cells/ml.
Cover each tube/flask with tinfoil to prevent photobleaching
(see Note 13).
15. To the splenocytes, add an equal volume of CFSE
high or CFSElow as follows:
(a) CFSE
high solution is added to “Target” cells to obtain a
final conc. of 2.5 μM CFSE pro-dye.
(b) CFSE
low solution is added to “Nontarget” cells to obtain
a final conc. of 0.25 μM CFSE pro-dye.
16. Gently agitate the cells for 8 min at room temperature (see
Note 14).
17. Add an equal volume of ice-cold FBS to each tube and place on
ice for 5 min (see Note 15).
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Felicity C. Stark et al.