18. Centrifuge cells for 5 min at 400 Â g, 4
C in a swinging bucket
rotor to pellet cells, then discard supernatant.
19. Separately resuspend “Target” and “Nontarget” cells in 10 ml
of HBSS. Take a 10 μl aliquot of each for cell counting.
20. Centrifuge cells for 5 min at 400 Â g, 4
C in a swinging bucket
rotor to pellet cells, then discard supernatant.
21. Separately resuspend “Target” and “Nontarget” cells in HBSS
to a final concentration of 1 Â 10
8 cells/ml.
22. Combine equal volumes of “Target” cells and “Nontarget”
cells and keep on ice. Take into consideration how many mice
will need to be injected. For instance, injecting 25 mice with
200 μl each requires a minimum volume of 6.0 ml (includes
extra 1.0 ml volume for syringe loading) (see Note 16).
23. Keeping the cells on ice at all times, inject 20 Â 10
6 cells in
200 μl intravenously per mouse (see Note 17).
3.2 Detection
of “Target”
and “Nontarget” Cells
in Mice
1. Euthanize all mice injected with target cells at approximately
20 h postinjection. Anesthetize the mice using isoflurane anesthesia, perform cervical dislocation and harvest the spleens (see
Note 18).
2. Process each spleen separately. Mash the spleen between the
frosted ends of two glass slides in 4 ml of R10.
3. Create a 96-well table to record where each spleen sample will
be aliquoted. Transfer 400 μl from each sample to a 96-well
deep-well plate (see Note 19).
4. Centrifuge cells for 5 min at 400 Â g, 4
C in a swinging bucket
rotor to pellet cells, then discard supernatant.
5. Resuspend cells in 200 μl of FACS buffer.
6. Filter the cell solution to remove clumps immediately before
acquisition by flow cytometry. Transfer the cell solution
through a Multiscreen 60 μm nylon mesh 96-well filter plate,
then aliquot the samples into a U bottom 96-well plate (see
Note 20).
7. Use a plate reader attachment, such as the High-Throughput
Sampler (HTS) configured to a BD LSR Fortessa™ flow cytometer, to acquire samples on a U-bottom 96-well plate. Take
care to ensure that CFSE positive cells (two peaks) are visible
on the FITC detector (Fig. 2d, h) (see Note 21).
8. Calculate the % CTL killing for each experimental mouse by
comparing the % “Target” cells of all CFSE
+ cells to the %
“Nontarget” cells of all CFSE
+ cells (T/NT) using the formula
shown in Fig. 3A.
The (T/NT)naı ¨ve would be the average of all naı ¨ve mice
used in the study, and the (T/NT)exp. would represent the
A Method to Evaluate In Vivo CD8
+ T Cell Cytotoxicity in a Murine Model
555
C in a swinging bucket
rotor to pellet cells, then discard supernatant.
19. Separately resuspend “Target” and “Nontarget” cells in 10 ml
of HBSS. Take a 10 μl aliquot of each for cell counting.
20. Centrifuge cells for 5 min at 400 Â g, 4
C in a swinging bucket
rotor to pellet cells, then discard supernatant.
21. Separately resuspend “Target” and “Nontarget” cells in HBSS
to a final concentration of 1 Â 10
8 cells/ml.
22. Combine equal volumes of “Target” cells and “Nontarget”
cells and keep on ice. Take into consideration how many mice
will need to be injected. For instance, injecting 25 mice with
200 μl each requires a minimum volume of 6.0 ml (includes
extra 1.0 ml volume for syringe loading) (see Note 16).
23. Keeping the cells on ice at all times, inject 20 Â 10
6 cells in
200 μl intravenously per mouse (see Note 17).
3.2 Detection
of “Target”
and “Nontarget” Cells
in Mice
1. Euthanize all mice injected with target cells at approximately
20 h postinjection. Anesthetize the mice using isoflurane anesthesia, perform cervical dislocation and harvest the spleens (see
Note 18).
2. Process each spleen separately. Mash the spleen between the
frosted ends of two glass slides in 4 ml of R10.
3. Create a 96-well table to record where each spleen sample will
be aliquoted. Transfer 400 μl from each sample to a 96-well
deep-well plate (see Note 19).
4. Centrifuge cells for 5 min at 400 Â g, 4
C in a swinging bucket
rotor to pellet cells, then discard supernatant.
5. Resuspend cells in 200 μl of FACS buffer.
6. Filter the cell solution to remove clumps immediately before
acquisition by flow cytometry. Transfer the cell solution
through a Multiscreen 60 μm nylon mesh 96-well filter plate,
then aliquot the samples into a U bottom 96-well plate (see
Note 20).
7. Use a plate reader attachment, such as the High-Throughput
Sampler (HTS) configured to a BD LSR Fortessa™ flow cytometer, to acquire samples on a U-bottom 96-well plate. Take
care to ensure that CFSE positive cells (two peaks) are visible
on the FITC detector (Fig. 2d, h) (see Note 21).
8. Calculate the % CTL killing for each experimental mouse by
comparing the % “Target” cells of all CFSE
+ cells to the %
“Nontarget” cells of all CFSE
+ cells (T/NT) using the formula
shown in Fig. 3A.
The (T/NT)naı ¨ve would be the average of all naı ¨ve mice
used in the study, and the (T/NT)exp. would represent the
A Method to Evaluate In Vivo CD8
+ T Cell Cytotoxicity in a Murine Model
555