solution is 0.5% and in our experience, this has little to no effect
on cell viability.
10. Depending on the peptide chosen, its concentration and incubation duration will vary. It is important to take into consideration whether the peptide will be soluble in a cell culture
solution by looking at the chemical and physical properties of
the amino acid sequence.
11. If using Falcon tubes, use a tube rack and keep the lids loosened. If using T75 flasks with ventilated caps, tighten the lid
and keep the flask upright.
12. Prepare a volume based on the number of cells. For instance,
suspend 500 Â 10
6 cells in 25 ml of room temperature PBS to a
concentration of 20 Â 10
6 cells/ml. A 25 ml 2Â solution of
CFSE would then be combined with the cells to obtain a final
concentration of 10 Â 10
6 cells/ml.
13. The CFSE pro-dye solution (CFDSE) is not fluorescent and
does not require protection from light, but once it is combined
with cells the resulting fluorescence can be negatively affected
by exposure to light. The following steps will include the rapid
addition of other buffers; ensure that there is enough room in
each tube for the addition of 4Â the existing volume. For
instance, if you have 20 ml of cells in a 50 ml Falcon tube,
divide it among two more 50 ml Falcon tubes so that there is
enough space for the subsequent steps. Be sure to transfer the
labels “Target” or “Nontarget”; there is no way to recover the
cells if a mixup occurs.
14. Since CFSE pro-dye rapidly stains cells, the cells and staining
solution are combined in equal volumes to ensure all cells are
exposed to CFSE pro-dye at the same concentration. Unequal
staining can not only affect cell viability (higher CFSE pro-dye
concentration is associated with cell death) but also the intensity of the fluorescent peaks, which can become wider and less
distinct and make it difficult to discern between the CFSE
high
and CFSE
low peaks. The duration of CFSE staining ranges
from 5 to 20 min according to published protocols. We obtain
consistent staining with 8 min of staining.
15. These steps should be completed quickly. FBS is added to stop
CFSE staining, also known as quenching the reaction. If CFSE
pro-dye is left too long with the cells, it can become toxic to the
cells and in some cases a slightly higher cell death is observed
for cells stained with CFSE
high solution. If this becomes an
issue, next time consider starting with slightly more cells for
the “Target” cell population in step 8.
16. Combining an equal proportion of “Target” and “Nontarget”
cells is ideal but exact equality is not required. “Target” cell
killing is calculated relative to “Nontarget” cells in the same
A Method to Evaluate In Vivo CD8
+ T Cell Cytotoxicity in a Murine Model
557
on cell viability.
10. Depending on the peptide chosen, its concentration and incubation duration will vary. It is important to take into consideration whether the peptide will be soluble in a cell culture
solution by looking at the chemical and physical properties of
the amino acid sequence.
11. If using Falcon tubes, use a tube rack and keep the lids loosened. If using T75 flasks with ventilated caps, tighten the lid
and keep the flask upright.
12. Prepare a volume based on the number of cells. For instance,
suspend 500 Â 10
6 cells in 25 ml of room temperature PBS to a
concentration of 20 Â 10
6 cells/ml. A 25 ml 2Â solution of
CFSE would then be combined with the cells to obtain a final
concentration of 10 Â 10
6 cells/ml.
13. The CFSE pro-dye solution (CFDSE) is not fluorescent and
does not require protection from light, but once it is combined
with cells the resulting fluorescence can be negatively affected
by exposure to light. The following steps will include the rapid
addition of other buffers; ensure that there is enough room in
each tube for the addition of 4Â the existing volume. For
instance, if you have 20 ml of cells in a 50 ml Falcon tube,
divide it among two more 50 ml Falcon tubes so that there is
enough space for the subsequent steps. Be sure to transfer the
labels “Target” or “Nontarget”; there is no way to recover the
cells if a mixup occurs.
14. Since CFSE pro-dye rapidly stains cells, the cells and staining
solution are combined in equal volumes to ensure all cells are
exposed to CFSE pro-dye at the same concentration. Unequal
staining can not only affect cell viability (higher CFSE pro-dye
concentration is associated with cell death) but also the intensity of the fluorescent peaks, which can become wider and less
distinct and make it difficult to discern between the CFSE
high
and CFSE
low peaks. The duration of CFSE staining ranges
from 5 to 20 min according to published protocols. We obtain
consistent staining with 8 min of staining.
15. These steps should be completed quickly. FBS is added to stop
CFSE staining, also known as quenching the reaction. If CFSE
pro-dye is left too long with the cells, it can become toxic to the
cells and in some cases a slightly higher cell death is observed
for cells stained with CFSE
high solution. If this becomes an
issue, next time consider starting with slightly more cells for
the “Target” cell population in step 8.
16. Combining an equal proportion of “Target” and “Nontarget”
cells is ideal but exact equality is not required. “Target” cell
killing is calculated relative to “Nontarget” cells in the same
A Method to Evaluate In Vivo CD8
+ T Cell Cytotoxicity in a Murine Model
557