Anti cd14 apc

Using a 7-color flow cytometry assay, 1×10⁶ fresh, frozen/not rested and frozen/rested overnight PBMC were analyzed for phenotype and the activation marker, CD69. To determine viability, PBMC were washed twice with PBS, resuspended and incubated in 1μL of Aqua amine-reactive viability dye (Molecular Probes, Inc. Eugene, OR) for 20 min at RT in the dark, washed and incubated with 10μL of Fc block (Miltenyi Biotec Inc., Auburn, CA) for another 20 min at RT in the dark. The cells were washed with FACS buffer (0.2% BSA+0.1% NaN₃ in PBS) and incubated for 20 min in the dark at 4°C with anti-CD3-eFluor 450, anti-CD19-APCeFluor 780 (both from eBioscience), anti-CD56-PE/Cy7, anti-CD14-APC, anti-CD16-FITC and anti-CD69-PE/Cy5 (Biolegend, San Diego, CA). After incubation, cells were washed in FACS buffer and fixed in 1% paraformaldehyde. Duplicate sets of PBMC: fresh, frozen/thawed and frozen/thawed/rested overnight, were incubated overnight with or without IL-2 and were stained with identical cocktails or in the absence of anti-CD69 (supplemental Figure 1) for fluorescence minus one (FMO) control (Supplemental Figure 1). Samples were analyzed using an LSRII flow cytometer and FlowJo software (TreeStar Inc., Ashland, OR). Dead cells were excluded from analysis by gating on Aqua amine-reactive viability dye negative cells.

Non-activated bbPBMCs seeded at 4 X 10⁶ were treated in vitro with the indicated ligands for 7 days in full RPMI containing 10% (v/v) cs-FCS, 2mM L-glutamine, 0.1 mg/mL sodium pyruvate, 100 IU/mL penicillin, 100 mg/mL streptomycin and 30 U/mL IL-2, while hPBMCs were thawed and rested overnight in full RPMI in an incubator at 37°C. Cells were washed with 1 X PBS containing 1% cs-FCS and subsequently stained with anti-human CD3 FITC (300306), anti-CD4 PE-DAZZLE 594 (357412), anti-CD8 PE/Cy5 (300910), anti-CD14 APC (325608), anti-CCR5 PE (359106), anti-CD69 PE/Cy7 (310912) and the viability dye, ZOMBIE NIR (423113) (Biolegend, USA), for 15 min in the dark at room temperature. Fluorescence minus-one (FMO) controls were used for gating as indicated in Supplementary Fig. S1. Cells were washed with 1 X PBS containing 1% cs-FCS then resuspended in 1 X Cell Fix solution (BD, USA) and analyzed using a LSRII flow cytometer (BD, USA) and FlowJo software version 10.1 (Treestar Inc., Ashland, Ore). Dead cells were excluded from the scatter plots prior to analysis and only single cellular populations were analyzed (Supplementary Fig. S1). Results were plotted as frequency (percentage of total), or expression as median fluorescence intensity (MFI) per number of double-positive cells.