Reagents:

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

BD Cytofix (554655)

2 mg/ml Collagenase A (Roche 11088793001)

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

Protocol:

Prepare single cell suspension from tissue (up to 1cm³) with 1 hour digestion using 2 mg/ml Collagenase A in 20 ml HBSS + 40 ul DNase (50 U/ml). Filter with 70 um.
Samples can be frozen in 90%FCS/10%DMSO or stained immediately (see below)
Wash samples and plate into a 96-well round bottom plate. Spin and remove supernatant.
Prepare a 1/500 dilution of Live/Dead Aqua stain in PBS (no protein) with 1/20 dilution of human Fc block (i.e. 2ul Live/Dead + 50ul Fc block per 1000ul PBS). Add 200ul to samples, mix, and incubate for 30 min on ice. Prepare antibody mixtures (see panel on right).
Spin down cells to remove supernatant. Add 200 ul of antibody mixture to cells and incubate for 30 min on ice.
Wash cells 1x 200ul FACS Buffer.
Fix cells with BD Cytofix for 30 mins.
Wash cells 1x FACS buffer.
Store cells at 4C.

12/2017

Human Flow Cytometry