Fc block cocktail

Cells were washed in 150 μL of BSA Stain Buffer (BD Biosciences) in preparation of phenotype staining. 50μL of Fc Block cocktail (composed of Fc Block [Biolegend], Live Dead Near IR Fixable Viability Stain (Thermo Fisher), BSA Stain Buffer) was added to each sample and incubated for 10 min at room temperature. After the incubation period, cells were stained with a monoclonal antibody cocktail containing CD19-PacBlue, CD45-PE, CD45RA-FITC, CD4-PerCP-Cy5.5, CD16-PE, CD56-PE, CD19-PE-Cy7, and CD3-APC. All antibodies were received from Biolegend and diluted at a 1:50 ratio in BSA Stain Buffer. Following a 30-min staining period at 4 °C, samples were washed by centrifuging at 400 x g for 5 min (room temperature) and resuspended in BSA Stain Buffer. Following the third centrifugation step, PBMCs were resuspended in BSA Stain Buffer and 16% PFA was added to each sample for a final concentration of 1.6% PFA. After a 10-min fixation period at 21 °C, cells were washed and resuspended in BSA Stain Buffer for flow cytometry acquisition.

Cells were transferred from the 96 well plate to a PCR plate (Thermo Fisher) and centrifuged at 400 x g for 5 min at room temperature, making sure to aspirate the resulting supernatant in between each transfer. Once all cells were transferred to the new PCR plate, cells were centrifuged and resuspended in FBS Stain Buffer (BD Biosciences). FC Block Cocktail (Human TruStain FcX Fc Receptor Blocking Solution [Biolegend] and FBS Stain Buffer) was added to each sample and incubated for 10 min at 21 °C. Following the blocking incubation, Live Stain Cocktail, composed of Live/Dead-Aqua (1:100, Biolegend), CD8-PerCP-Cy5.5 (1:20, Biolegend), and CD14-PE-Dazzle594 (1:20, Biolegend) in FBS Stain Buffer, was added to each well, and the plate was incubated for a subsequent 30 min at 21 °C. Samples were washed twice via centrifugation and resuspension in 150 μL of FBS Stain Buffer, and finally resuspended in 60 μL of FBS Stain Buffer. 16% PFA was added to each sample for a final concentration of 1.6% PFA and incubated for 10 min at 21 °C to fix the cells. After fixation, cells were washed as before and resuspended in FBS Stain Buffer in preparation for intracellular staining.