Fcr cd16 32 blocking reagent

PB and BM samples from HDs and AA patients were collected in EDTA anticoagulant tubes and stored at 4 °C. PBMCs and BMMCs were isolated by density gradient centrifugation using Ficoll‐Paque PLUS within 24 h. PBMCs and BMMCs were blocked using FcR (CD16/32) Blocking Reagent (Miltenyi), and stained with the following antibodies/reagents: PE anti‐human CD158f (KIR2DL5) antibody (Biolegend), PE anti‐human CD158e1 (KIR3DL1, NKB1) antibody, PE mouse anti‐Human CD158a (BD Pharmingen), PE mouse anti‐human CD158b (BD Pharmingen), APC anti‐human TCRγ/δ (Biolegend), Biotin anti‐humanTCRvδ2 (Biolegend), APC/Cyanine7 streptavidin (Biolegend), human KIR3DL2/CD158k PE‐conjugated antibody (R&D), anti‐humanCD261(DR4)‐APC (Miltenyi), FITC anti‐human CD8a antibody (Biolegend), APC‐Cy7 anti‐human CD4 antibody (Biolegend), Percp‐Cy5.5 anti‐human CD25 antibody (Biolegend), PE‐Cy7 anti‐human CD127 antibody (eBioscience), PE/Cyanine7 Streptavidin (Biolegend), PerCP/Cyanine5.5 anti‐human CD19 (BioLegend), PE‐Cy7 anti‐human CD3 antibody (BioLegend), APC anti‐human TCRγ/δ antibody (BioLegend), and PE anti‐human CD184 (CXCR4) antibody (BioLegend). The cells were resuspended in 400 µl 0.1 µg mL⁻¹ DAPI solution (Salarbio). Treg cells (CD8a⁻CD4⁺CD127^−/lowCD25⁺) were sorted using an Arial II (BD Biosciences).

Venous blood samples from healthy donors were collected in EDTA anticoagulant tubes and stored at 4°C. PBMCs were isolated by density gradient centrifugation using Ficoll–Paque PLUS (Cat No. 17-1440-03, GE Healthcare). PBMCs were blocked by FcR (CD16/32) Blocking Reagent (Cat No. 130-059-901, Miltenyi Biotec) antibody, and stained with the following antibodies: APC/Cyanine7 anti-human CD4 antibody (A161A1, Cat No. 357415, Biolegend), FITC anti-human CD8a antibody (RPA-T8, Cat No. 301050, Biolegend), PE-Cyanine 7 anti-human CD127 antibody (A7R34, Cat No. 25-1271-82, eBioscience), PerCP/Cyanine5.5 anti-human CD25 antibody (BC96, Cat No. 302626, Biolegend), and APC anti-human CD30 antibody (BY88, Cat No. 333910, Biolegend). Finally, the cells were resuspended in 400 ul 0.1 ug/ml DAPI solution (Cat No. C0060, Salarbio) and analyzed using an Arial II cytometer (BD Biosciences). Flow cytometry data were analyzed using FlowJo (Three Star, Ashland OR). Percentage data were presented as mean ±  SD using GraphPad Prism.