Percp cyanine5.5 anti human cd19

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).

We used a PBMC isolation kit (Solarbio, P8900, Shanghai, China) to isolate PBMCs. First, in order to detect antigen-specific SARS-CoV-2 B cells, approximately 1.0 × 10⁶ PBMCs were treated with LIVE/Dead Zombie Aqua Dye (423102, BioLegend; Beijing, China, 1 μL/well) at 4 °C for 15 min. Biotinylated SARS-CoV-2 RBD protein was combined with Streptavidin-BV421 (405225, BioLegend) at a 4:1 molar ratio and let to sit for one hour at 4 °C to obtain the RBD probe. Fluorescence-Activated Cell Sorting (FACS) buffer (PBS + 2% FBS) was used to resuscitate PBMCs into a cell suspension, then the sample was transferred to a microplate, ensuring 100 μL per well. Then, at 4 °C, antigen probes with a concentration of 1.5 μL per well were used for staining for 30 min, followed by these conjugated antibodies: APC anti-human CD3 (300412, BioLegend; 1 μL/well), PerCP/Cyanine 5.5 anti-human CD19 (302230, BioLegend; 1 μL/well), FITC anti-human CD21 (354910, BioLegend; 1 μL/well), PE anti-human CD27 (302808, BioLegend; 1 μL/well). After being stained, the cells were washed and resuspended in 200 μL FACS buffer. The full gating strategy is illustrated in Supplementary Figure S3A.