Cd3 okt3

At each WIHS visit, PBMCs were isolated from venous blood by standardized methods, frozen, and stored in liquid nitrogen in a specimen repository. Cryopreserved PBMCs stored in liquid nitrogen were warmed in 37°C water bath, then removed and immediately diluted with 1 mL of warm cRPMI and transferred and diluted again in an additional 8mL of warm cRPMI. Samples were then washed with PBS and stained with viability reagent (Ghost Dye^TM Red 710, Tonbo Bioscience) according to manufacturer’s recommendation. Samples were stained with antibody cocktail containing CD14 (M5E2, Biolegend), CD16 (3G8, Biolegend), and dump channel: CD3 (OKT3, Tonbo Bioscience), CD19 (HIB19, Tonbo Bioscience), CD56 (5.1H11, Biolegend), CD66b (G10F5, Biolegend), gated based on living cells and excluding dump channel-positive cells. Intermediate monocytes were defined as CD14⁺CD16⁺ and non-classical monocytes were defined as CD14^dimCD16⁺. Monocytes were sorted directly into TRIzol® LS Reagent (Life Technologies) and frozen at -80°C. FCS files were exported from FACS Diva (BD Bioscience) and processed using FlowJo v10.2 (FlowJo, LLC). The quantification of intermediate and non-classical monocytes are estimated from the FACS data from all PBMC samples.