Pe anti human cd16 antibody

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.

Approximately 10⁶ HL60 cells per treatment were incubated with 5 µL of mouse Phycoerythrin (PE) anti-human CD16 antibody (#360703; BioLegend, San Diego, CA, USA) for 30 min at RT in the dark, and then, cells were washed with 1 mL 10 mM PBS + 0.5% BSA+ 0.065% sodium azide. All samples were analyzed on a FACScalibur flow cytometer equipped with CellQuest Pro software (BD Biosciences) and FlowJo v10 software (BD Biosciences). The CD16 binding-capacity and thus cell differentiation were measured in comparison with DMSO-induced HL60 cells without incubation with CD16 antibody (Figure S1).

PE mouse anti-human IgG and APC-H7 mouse anti-human CD3 were purchased from BD (Franklin Lakes, NJ, USA). Goat anti-human IgG1 PE was purchased from Thermo Fisher Scientific (Waltham, MA, USA). PE/Cyanine7 anti-human IFN-γ antibody, PE anti-human TNF-α antibody, APC anti-human CD107a (LAMP-1) antibody, APC anti-human CD56 (NCAM) antibody, PE anti-human CD16 antibody, PE anti-human CD335 (NKp46) antibody (9E2), Brilliant Violet 421™ anti-human CD45 antibody, PE/Cyanine7 anti-human CD14 antibody, PE anti-human CD19 antibody, and Zombie NIR™ Fixable Viability Kit were purchased from Biolegend (San Diego, CA, USA).