Apc cyanine7 anti human cd3 antibody

Cryopreserved CD3+ Pan T cells (PB009-1F-C-5M), CD19+ B cells (PB010-P-F-C), CD14+ monocytes (PB-011-P-F-1-C), and CD56+ natural killer cells (PB012-P-F-C) were purchased from AllCells (Shanghai, China). The CD3+ Pan T cells were stained with APC/Cyanine7 anti-human CD3 Antibody (300425, BioLegend), FITC anti-human CD4 Antibody (300506, BioLegend) and PE anti-human CD8 Antibody (344705, BioLegend) following the manufacturer’s instructions. Then, the Pan T cells were sorted into CD4+ T cells and CD8+ T cells with a BD FACSAria III instrument. CD14+ monocytes were stained with PE anti-human CD14 Antibody (301805, BioLegend) and APC anti-human CD16 Antibody (302011, BioLegend) following the manufacturer’s instructions. CD14+ monocytes were further separated into CD16+ monocytes and CD14+ monocytes (CD16-) with a BD FACSAria III instrument.
For fresh PBMCs, venous blood was collected from a healthy donor into a plastic blood tube spray-coated with K₂EDTA (367863, Becton Dickinson, NJ, USA). Blood was dissolved in an equal volume of 1 x DPBS solution and added to a SepMate™-15 tube (86415, StemCell Technologies, Canada) containing Histopaque-1077 (10771-100 ml, Sigma-Aldrich, MO, USA). After centrifugation (1200 x g, 10 min), PBMCs were collected, washed twice, and resuspended with a 1 x DPBS solution.

PBMCs with or without mitogen treatment and MSC co-culture were harvested and pooled from the 96-well plates at the times specified; 1 × 10⁶ cells were centrifuged at 600 × g for 6 min, resuspended in 100 µL of HBSS containing 5 µL of Human TruStain FcX™, and incubated for 5 min at room temperature. PBMCs were then stained with the following markers: 2.5 µL of APC/Cyanine7 anti-human CD3 Antibody (BioLegend cat # 300318, RRID : AB_314054, San Diego, CA) and 2 µL of bovine lactadherin (Prolytix Cat# BLAC-FITC, Essence Junction, VT) with or without 2.5 µL of Brilliant Violet 421™ anti-human CD4 antibody (BioLegend Cat #357424, RRID : AB_2721519, San Diego, CA). Cells were stained for 15 min at room temperature in the dark followed by a 5-min incubation using 5 µL of 7AAD (BioLegend, San Diego, CA). HBSS (100 µL) was added to the samples before analysis on a BD FACS Canto II flow cytometer (BD Biosciences, Franklin Lakes, NJ). Data are expressed as % positive cells of the live population. Suppression was calculated as above.

Fresh blood samples at indicated time points were collected for flow-cytometry analysis. Circulating numbers of CD4⁺ T cells, CD8⁺ T cells, CD3^-CD16⁺CD56⁺ NK cells, and CD19⁺ B cells were determined by using TruCOUNT tubes and BD Multitest 6-color TBNK Reagent Kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions as previously described (Luo et al, 2021 (link)). For lymphocyte subset analysis, following antibodies were used: PerCP/Cyanine5.5 anti-human CD45 Antibody (Biolegend, 304028), FITC anti-human CD3 antibody (BD Biosciences, 561802), PE/Cyanine7 anti-Human CD4 (BD Biosciences, 560649), APC/Cyanine7 anti-Human CD8 (BD Biosciences, 557834), APC anti-human CD19 Antibody (Biolegend, 302212), PE anti-human CD16 Antibody (Biolegend, 302056), PE anti-human CD56 Antibody (Biolegend, 318306), FITC anti-Human CD38 (BD Biosciences, 567147), PerCP/Cyanine5.5 anti-Human CD27 (BD Biosciences, 560612). For CAR T-cell percentage analysis, PerCP anti-Human CD45 (BD Biosciences, 347464), APC/Cyanine7 anti-human CD3 Antibody (Biolegend, 344818) and FITC-labeled human BCMA Fc tag protein (Acrobiosystems, BCA-HF254) were used. All antibodies were used at the manufacturer’s recommended concentration. FlowJo v10 was used for analysis.