Percp anti human cd14

Flow cytometric analyses were conducted to supplement the quantification of the effects of HCQ and QC on TNF-α obtained through ELISA. PBMCs were isolated and treated with HCQ and QC, but not a combination of both, according to the procedure delineated. They were subsequently incubated for 3.5 hours at 37 °C and 5% CO₂. BD GolgiPlug protein transport inhibitor containing brefeldin A (BD Biosciences) was added to the LPS-stimulated PBMCs 40 minutes after stimulation. To detect intracellular expression of TNF-α in mDCs and monocytes, PBMCs were stained with FITC lineage 1 cocktail (containing anti-human antibodies against CD3, CD16, CD19, CD20, and CD56) (BioLegend, San Diego, CA), APC/Cy7 HLA-DR (BioLegend), PE anti-human CD11c (BioLegend), and PerCP anti-human CD14 (BioLegend) before being set on ice for 25 minutes, treated with fixation and permeabilization solution (BD Cytofix/Cytoperm Plus, BD Biosciences) for 20 minutes, washed (BD PermWash, BD Biosciences), and stained with allophycocyanin mouse anti-human TNF-α (BD Biosciences) for an additional 20 minutes. Cells were analyzed with an LSRII flow cytometer (Becton Dickinson, San Jose, CA) and FloJo software (Tree Star, Ashland, OR) at the Flow Cytometry and Cell Sorting Core of the Abramson Cancer Center of the University of Pennsylvania. A total of 150,000 events of live cells were acquired.