Anti his pe

Cryopreserved 10⁷ PBMC were thawed into 1 ml preheated RPMI1640, centrifuged at 300 × g for 5 min, resuspended in 500 μl FACS buffer (PBS + 2% FBS), and incubated with 200 nM his-tagged antigen (gH/gL) for 45 min at 4 °C. The PBMC were then washed two times with 1 ml FACS buffer and resuspended in 100 μl FACS buffer. The PBMC were stained with the following antibodies: CD3-PE-Cy5 (BD Biosciences Cat#555341) at a 1:25 dilution, CD14-PE-Cy5 (eBioscience Cat#15-0149-42) at a 1:50 dilution, CD16-PE-Cy5 (BD Biosciences Cat#555408) at a 1:25 dilution, CD235a-PE-Cy5 (BD Biosciences Cat#559944) at a 1:100 dilution, CD19-APC-Cy7 (BD Biosciences Cat#348794) at a 1:100 dilution, CD20-PE-Cy7 (BD Biosciences Cat#335793) at a 1:200 dilution, IgG-FITC (BD Biosciences Cat#555786) at a 1:25 dilution, and anti-his-PE (BioLegend Cat#362603) at a 1:20 dilution for 30 min at 4 °C. The PBMC were washed three times with 1 ml FACS buffer and resuspended in 500 μl FACS buffer, then subjected to FACS on a BD FACS Aria II (BD Biosciences).
Antigen-positive B cells (CD3-, CD14-, CD16-, CD235a-, CD19+, CD20+, IgG+, PE+) were sorted individually into 96-well PCR vital-plates containing 20 μl first strand buffer (5 μl first strand buffer, 0.5 μl of RNase inhibitor (Invitrogen Cat#10777019), 1.25 μl of 100 μM DTT, 0.06 μl of IGEPAL (Sigma Cat#I8896).

Flow cytometry of PBMCs from one healthy donor with five times of vaccinations were conducted following methods described previously^{47 (link)}. All collections were conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of the Ethics Committee of Huashan Hospital (2021-749). This participant provided written informed consent. Briefly, PBMCs were stained with LIVE/DEAD Fixable Yellow Dead Cell Stain Kit (Invitrogen) at ambient temperature for 20 min, followed by washing with RPMI-1640 complete medium and incubation with 10 μg/mL SARS-CoV-2 Omicron BA.1- and MERS-CoV-S trimers with His tag at 4 °C for 1 h. Afterward, the cells were washed again and incubated with a cocktail of flow cytometry antibodies, containing CD3 APC-H7 (BD Biosciences), CD19 BV421 (BD Biosciences), CD27 APC (BD Biosciences), and anti-His PE (Biolegend), at 4 °C for 1 h. Stained cells were then washed, resuspended in RPMI-1640 complete medium and sorted for S trimer-specific memory B cells (CD3⁻CD19⁺CD27⁺S trimer⁺ live single lymphocytes). The sorted cells were loaded into the BD Rhapsody single-cell analysis platform, which could effectively capture and separate single cells. The BCR library preparation and quality control were performed according to the manufacturer’s protocol and sequenced on the NovaSeq PE150 platform (Illumina).

S2P spike trimer-specific memory B cells were isolated and sequenced using the protocol previously described by Liu et al.²¹ (link)^,27 (link) In brief, peripheral blood mononuclear cells (PBMCs) from patient 12 were stained with LIVE/DEAD Fixable Yellow Dead Cell Stain Kit (Invitrogen) at room temperature for 20 min, washed with RPMI-1640 complete medium, and incubated with 10 μg/mL B.1.351 S2P spike trimer at 4 °C for 45 min. Cells were then washed and incubated with a cocktail of flow cytometry and hashtag antibodies, containing CD3 PE-CF594 (BD Biosciences), CD19 PE-Cy7 (Biolegend), CD20 APC-Cy7 (Biolegend), IgM V450 (BD Biosciences), CD27 PerCP-Cy5.5 (BD Biosciences), anti-His PE (Biolegend), and human Hashtag 3 (Biolegend) at 4 °C for 1 h. Cells were then washed again, resuspended in RPMI-1640 complete medium, and sorted for S2P spike trimer-specific memory B cells (CD3−CD19+CD27+S trimer+ live single lymphocytes). These sorted cells were mixed with PBMCs from the same donor, labeled with Hashtag 1, and loaded to a 10X Chromium chip for the 5′ Single Cell Immune Profiling Assay (10X Genomics) at the Columbia University Human Immune Monitoring Core (HIMC; RRID:SCR_016740). Library prep and quality control were performed according to the manufacturer’s instructions and then sequenced on a NextSeq 500 (Illumina).