3. RPMI 1640 buffer: RPMI 1640 (Life Technologies, Carlsbad,
CA).
4. R10: RPMI containing 10% heat-inactivated fetal bovine
serum (FBS) and 2 mM L-glutamine.
5. PBS (pH 7.4), kept at room temperature (20–25
C) without
Ca
2+ or Mg
2+ (see Note 4).
6. FACS buffer: 2% FBS and 3 mM EDTA in PBS (see Note 5).
7. HBSS: Hank’s balanced salt solution (Thermofisher, Waltham,
MA, USA).
8. Dimethyl sulfoxide (DMSO), sterile (MilliporeSigma, Burlington, MA, USA).
9. T75 flasks.
10. Petri dishes.
11. Microcentrifuge tubes, 1.5 ml.
12. Falcon™ conical centrifuge tubes, 50 ml.
13. Round-bottom polystyrene tubes, 5 ml.
14. 96-Well DeepWell™ polypropylene microplates, 2 ml.
15. Multiscreen 60 μm nylon mesh Â96-well filter plate (MilliporeSigma, Burlington, Massachusetts).
16. U bottom Â96-well plate.
17. Carboxyfluorescein diacetate N-hydroxysuccinimidyl ester
(CFDSE), a cell-permeant non-fluorescent pro-dye that
becomes cleaved intracellularly to the membrane impermeant
fluorescent dye carboxyfluorescein succinimidyl ester (CFSE).
18. BD LSR Fortessa™ flow cytometer, or other flow cytometer
with a Blue laser (488 nm) and a FITC detector for CFSE’s
peak emission of 521 nm.
3 Method
3.1 Preparation
of Target Cells
1. Spleens are collected from C57BL/6 donor mice. Ten donor
spleens should be collected for every ~25 mice receiving
injected cells.
2. Anesthetize the mice using isoflurane and perform cervical
dislocation, then harvest the spleens (see Note 6).
3. Aseptically mash pooled spleens in a petri dish in R10 between
the frosted ends of two glass slides to obtain a single-cell
suspension. Use approximately 40 ml of R10 for every ten
donor spleens (see Note 7).
4. Filter splenocytes through a 75 μm cell strainer into a new
50 ml Falcon tube.
5. Centrifuge cells for 5 min at 400 Â g, 4
C in a swinging bucket
rotor to pellet cells, then discard supernatant.
A Method to Evaluate In Vivo CD8
+ T Cell Cytotoxicity in a Murine Model
553
CA).
4. R10: RPMI containing 10% heat-inactivated fetal bovine
serum (FBS) and 2 mM L-glutamine.
5. PBS (pH 7.4), kept at room temperature (20–25
C) without
Ca
2+ or Mg
2+ (see Note 4).
6. FACS buffer: 2% FBS and 3 mM EDTA in PBS (see Note 5).
7. HBSS: Hank’s balanced salt solution (Thermofisher, Waltham,
MA, USA).
8. Dimethyl sulfoxide (DMSO), sterile (MilliporeSigma, Burlington, MA, USA).
9. T75 flasks.
10. Petri dishes.
11. Microcentrifuge tubes, 1.5 ml.
12. Falcon™ conical centrifuge tubes, 50 ml.
13. Round-bottom polystyrene tubes, 5 ml.
14. 96-Well DeepWell™ polypropylene microplates, 2 ml.
15. Multiscreen 60 μm nylon mesh Â96-well filter plate (MilliporeSigma, Burlington, Massachusetts).
16. U bottom Â96-well plate.
17. Carboxyfluorescein diacetate N-hydroxysuccinimidyl ester
(CFDSE), a cell-permeant non-fluorescent pro-dye that
becomes cleaved intracellularly to the membrane impermeant
fluorescent dye carboxyfluorescein succinimidyl ester (CFSE).
18. BD LSR Fortessa™ flow cytometer, or other flow cytometer
with a Blue laser (488 nm) and a FITC detector for CFSE’s
peak emission of 521 nm.
3 Method
3.1 Preparation
of Target Cells
1. Spleens are collected from C57BL/6 donor mice. Ten donor
spleens should be collected for every ~25 mice receiving
injected cells.
2. Anesthetize the mice using isoflurane and perform cervical
dislocation, then harvest the spleens (see Note 6).
3. Aseptically mash pooled spleens in a petri dish in R10 between
the frosted ends of two glass slides to obtain a single-cell
suspension. Use approximately 40 ml of R10 for every ten
donor spleens (see Note 7).
4. Filter splenocytes through a 75 μm cell strainer into a new
50 ml Falcon tube.
5. Centrifuge cells for 5 min at 400 Â g, 4
C in a swinging bucket
rotor to pellet cells, then discard supernatant.
A Method to Evaluate In Vivo CD8
+ T Cell Cytotoxicity in a Murine Model
553