Anti human cd4 bv650

Frequencies of B cells, CD4⁺ T cells, and CD8⁺ T cells in human PBMC were determined by flow cytometry, as previously described [8 (link)]. Briefly, human PBMC with or without T or B cell depletion were stained with anti-human CD3-BV421 (BD Biosciences, clone UCHT1), anti-human CD4-BV650 (BD Biosciences, clone 2RPA-T4), anti-human CD8-APC (BD Biosciences, clone SK1), anti-human CD20-Percp/cy5.5 (BD Biosciences, clone 2H7), and anti-human CD45-FITC (BD Biosciences, clone 2D1) and measured by using LSR II flow cytometer (BD, USA). The data were analyzed using the FACS Express software (De Novo Software, USA, version 5).

Cryopreserved PBMCs were thawed, washed, and stained with Live/Dead Aqua Blue (Life Technologies), followed by surface staining with antihuman CD3 BV605, anti-human CD4 BV650, anti-human CD8 BV711, anti-human CD27 PerCP-eF710, CD45RO PE-CF594 (BD Biosciences), anti-human CD98 PE (BD Biosciences), anti-human CD71 APC, anti-human PD-1 PE-Cy7, and anti-human T-Bet BV421 (all from BioLegend unless otherwise noted). Anti-human CD14, anti-human CD16, and anti-human CD19 all tagged with V500 (BD Biosciences) were added in dump. For median fluorescence index determination, manual target channeling was performed with 1× beads (SpiroTech). Samples were run on modified 5 laser BD LSR Fortessa X-20. Data were analyzed on FlowJo (FlowJo).