Fitc conjugated anti cd16

Peripheral blood was extracted from subjects in EDTA (ethylene diamine tetraacetic acid) tubes and then 150 μL was pipetted into respective polypropylene tubes. Negative controls only consisted of tested blood without the addition of diagnostic antibodies. ACK (Ammonium Chloride-Potassium) Lysis buffer was used to lyse red blood cells. Cells were resuspended in FACS (fluorescence-activated cell sorting) buffer at a concentration of 1 x 10⁶ cells/ml and stained with 5 μL Zombie Aqua™ Fixable Viability (Biolegend, 423101), 5 μL FITC-conjugated anti-CD16 (BioLegend, 302006), and 5 μL APC-conjugated anti-CD294 (CRTH2) (Biolegend, 350109). Finally, the cells were harvested and resuspended in 500 μL PBS. Flow cytometry was performed including live/dead viability gate with CRTH2+ eosinophils identified as SSC^high, CD16- and CRTH2+ granulocytes. Isotype antibody controls were used to develop the gating and data analysis strategies. Flow cytometry was performed on BD FACSAria III. The data were analyzed with FlowJo.

Fc receptors were blocked with Fcγ blocker (BioLegend, San Diego, CA, USA), and the surface markers were stained with BV450-conjugated anti-CD3 (BioLegend, clone:UCHT1), FITC-conjugated anti-CD19 (BioLegend, clone: HIB19), APC/Cy7-conjugated anti-CD20 (BioLegend, clone: 2H7), PE-conjugated anti-CD14 (BioLegend, clone: ΦM P9), PE-conjugated anti-CD11c (BioLegend, clone: B-ly6), PerCP/Cy5.5-conjugated anti-CD56 (BioLegend, clone: B159), FITC-conjugated anti-CD16 (BioLegend, clone:3G8), PE-conjugated anti-CD123 (BioLegend, clone: 6H6), and APC/Cy7-conjugated anti-CD4 (BioLegend, clone: RPA-T4). After fixing and permeabilization, IGBP1 was stained with Alexa 647-conjugated anti-IGBP1 (Novus Biologicals, Centennial, CO, USA).