121
C for 15 min. Allow to cool to room temperature
(RT) before use. Prepare fresh each time.
3. Blood agar plates.
4. Cell culture flask with vented caps.
5. 10 μL sterile disposable inoculation loops.
6. Research CO 2 incubator.
7. Benchtop centrifuge.
8. Dulbecco’s phosphate buffered saline (DPBS).
9. Bacterial fixing solution: 0.5% formaldehyde solution in DPBS.
10. 0.1 M sodium hydrogen carbonate (0.1 M NaHCO 3 ): Dissolve 3.36 g of NaHCO 3 crystals in 396.64 mL of deionized
water. Sterilize at 121
C for 15 min. Allow to cool to room
temperature (RT) before use.
11. Spectrophotometer.
12. Cell staining solution: Dissolve 100 mg of 5(6)carboxyfluorescein N-hydroxysuccinimide ester (FAM SE) in
10 mL of dimethyl sulfoxide (DMSO) to give a final concentration of 10 mg/mL. Protect from light by wrapping in
aluminum foil.
2.2 ICA
1. Frozen stocks of PBMCs.
2. 5 mL round bottom polystyrene tubes.
3. Water bath.
4. Cell counter, slides, and trypan blue dye.
5. Single-color compensation beads.
6. RPMI-1640 medium.
7. R10 medium: RPMI-1640, 10% fetal bovine serum.
8. ICA buffer (DPBS, 1% bovine serum albumin, 50 U/mL
benzonase nuclease): Dissolve 5 g of bovine serum albumin
(BSA) flakes in 495 mL DPBS. Add 100 μL benzonase nuclease
to give 50 U/mL final concentration (see Note 1). Prepare on
day of experiment.
9. Zombie NIR™ Fixable Viability Kit: Follow manufacturer
instructions for preparation of the dye.
10. Anti-human CD45 streptavidin conjugate: Prepare using a
conjugation kit. Follow the manufacturer’s instructions (see
Note 2).
11. Biotin-SP-AffiniPure F(ab
0 )2 fragment goat anti-human IgG,
Fcγ fragment specific (biotin anti-human IgG).
12. Fluorescent antibodies:
(a) APC anti-human CD3 antibody (APC).
12
Sara Siris et al.
C for 15 min. Allow to cool to room temperature
(RT) before use. Prepare fresh each time.
3. Blood agar plates.
4. Cell culture flask with vented caps.
5. 10 μL sterile disposable inoculation loops.
6. Research CO 2 incubator.
7. Benchtop centrifuge.
8. Dulbecco’s phosphate buffered saline (DPBS).
9. Bacterial fixing solution: 0.5% formaldehyde solution in DPBS.
10. 0.1 M sodium hydrogen carbonate (0.1 M NaHCO 3 ): Dissolve 3.36 g of NaHCO 3 crystals in 396.64 mL of deionized
water. Sterilize at 121
C for 15 min. Allow to cool to room
temperature (RT) before use.
11. Spectrophotometer.
12. Cell staining solution: Dissolve 100 mg of 5(6)carboxyfluorescein N-hydroxysuccinimide ester (FAM SE) in
10 mL of dimethyl sulfoxide (DMSO) to give a final concentration of 10 mg/mL. Protect from light by wrapping in
aluminum foil.
2.2 ICA
1. Frozen stocks of PBMCs.
2. 5 mL round bottom polystyrene tubes.
3. Water bath.
4. Cell counter, slides, and trypan blue dye.
5. Single-color compensation beads.
6. RPMI-1640 medium.
7. R10 medium: RPMI-1640, 10% fetal bovine serum.
8. ICA buffer (DPBS, 1% bovine serum albumin, 50 U/mL
benzonase nuclease): Dissolve 5 g of bovine serum albumin
(BSA) flakes in 495 mL DPBS. Add 100 μL benzonase nuclease
to give 50 U/mL final concentration (see Note 1). Prepare on
day of experiment.
9. Zombie NIR™ Fixable Viability Kit: Follow manufacturer
instructions for preparation of the dye.
10. Anti-human CD45 streptavidin conjugate: Prepare using a
conjugation kit. Follow the manufacturer’s instructions (see
Note 2).
11. Biotin-SP-AffiniPure F(ab
0 )2 fragment goat anti-human IgG,
Fcγ fragment specific (biotin anti-human IgG).
12. Fluorescent antibodies:
(a) APC anti-human CD3 antibody (APC).
12
Sara Siris et al.