Anti human cd3 apc h7 clone sk7

First, we obtained 100 ml peripheral blood from a healthy donor who had received and passed the screening of infectious diseases, including hepatitis B, hepatitis C, syphilis, and HIV. Human PBMCs were isolated using Ficoll–Paque-based density gradient centrifugation. Next, we induced and extended the population of Vγ9Vδ2 T cells using our patented formula (Patent No. ZL201811580040.2). PBMCs at a density of 3 × 10⁶ cells/ml were cultured in RPMI 1640 (Gibco) containing 10% FBS and 1% penicillin–streptomycin and supplemented with 50 μM zoledronic acid (Sigma), 100 IU/ml recombinant human interleukin (IL)-2 (Beijing Four Rings Bio-Pharm Co.), 100 IU/ml recombinant human IL-15 (Peprotech), and 70 μM L-ascorbic acid (Sigma) on day 0. Afterwards, fresh medium and cytokines were replaced every 2–3 days. After being cultured for approximately 10 days, the viability of Vγ9Vδ2 T cells was determined using flow cytometry utilizing the PE-annexin V apoptosis kit (BD Biosciences, San Jose, CA, USA). In addition, Vγ9Vδ2 T cells were stained using anti-human CD3-APC-H7 (clone SK7; BD Biosciences), anti-human TCR Vδ2-Percp (clone B6; Biolegend), and anti-human CD314 (NKG2D)-PerCP/Cy5.5 (clone 1D11; BD Biosciences) antibodies to identify the cell phenotypes.

Thawed cells were stained with diluted LIVE/DEAD discrimination dye and subsequently stained for 15 minutes at room temperature with titrated amounts of anti-human CD3 Cy7-H7, anti-human CD4 PE-Cy5.5, and anti-human CD45RO ECD. Cells were washed once with 1X DPBS and resuspended in complete RPMI for sorting. Stained cells were sorted for live memory CD4 T cells using a BD S6 sorter the gating scheme: lymphocytes, singlets, live CD3+ cells, CD4+CD45RO+ cells.
Antibodies & Dyes: Anti-human CD3 APC-H7, clone SK7 –BD Biosciences–Cat#: 641397–1:30 dilution; Anti-human CD4 PE-Cy5.5, clone S3.5 –ThermoFisher Scientific–Cat#: MHCD0418–1:200 dilution; Anti-human CD45RO ECD, clone UCHL1 –Beckman Coulter–Cat# IM2712U – 1:30 dilution; LIVE/DEAD Fixable Violet Dead Cell Stain Kit, for 405nm excitation–ThermoFisher Scientific–Cat#: L34955–1:40 Dilution.