Buffers:

FACS Buffer: 500 mls PBS + 1 ml EDTA (0.5M stock) + 5 ml BSA (100 mg/ml stock)

RBC Lysis Buffer: 10x stock (8.02 g NH₄Cl, 0.84 g NaHCO₃, 0.29 g EDTA [no sodium], 100 ml milliQ); use 1 ml per 9 ml milliQ

BD Cytofix (554655)

2 mg/ml Collagenase A (Roche 11088793001)

DNase I (Roche 10104159001;100mg @ 2000U/mg): Prepare stock at 25 U/ul

Protocol:

Perfuse mouse with 10 mls of PBS/Heparin. Harvest tumors and mince in petri dish. Transfer to 50 ml pyrex container.
30 min digestion with 2 mg/ml Collagenase A in 20ml DMEM + 80ul DNase (50 U/ml) with mild agitation at 35°C. Filter with 100um. Count and transfer 2x10⁶ cells per well. Note that Collagenase A cleaves CD4; digestion over 30 min is not recommended. Stop reaction with 200 ul of 0.5M EDTA if tissue preparation will be over 30 min.
Warm up Live-Dead Aqua stain to room temperature and prepare a 1/500 dilution in PBS only. Use this to dilute Fc block 1/500, then add 200 ul per well. Mix and incubate for 30 min on ice.
During Fc blocking step, prepare antibody mixtures. Spin down cells and remove supernatant.
Add 200ul of antibody mixture to cells and incubate for 30 min on ice. Wash 1x 200 ul FACS Buffer
Fix cells for 30 min with 100 ul BD Cytofix
Wash cells 1x 200 ul FACS Buffer
Add 200 ul FACS Buffer. Store cells in the dark at 4°C until the run. Ensure plates or tubes are sealed to prevent evaporation.
1. Fixed cells should be stable for up to 1 week prior to analysis.
2. Conjugated antibodies (PE-Cy7, APC-Cy7) will degrade over time once exposed to fixative.

For Intracellular Staining:

After fixation with BD Cytofix, remove supernatant, then incubate with working dilution of Cytoperm Buffer (10x stock 561651) for 20-30 min.
Remove supernatant and add intracellular antibody appropriately diluted in 1x Cytoperm for 30 min.
Wash 1x with 1x Cytoperm.
Add 200 ul FACS Buffer and store sample until analysis.

12/2017

Mouse Flow Cytometry