Ab52192

B cells isolated from human PBMCs by Magnetic-Activated Cell Sorting (17954, StemCell Technologies) were immobilized onto glass slides coated with poly-L-lysine (Sigma-Aldrich). The BCR was labeled with a human IgM+G antibody (Alexa Fluro 546-(Fab)2-anti-Ig(M+G)). This antibody was generated from the F(ab′)2 fragment (Jackson ImmunoResearch, West Grove, PA) using a published protocol (23 (link)). Alexa Fluro 546-(Fab)2-anti-Ig(M+G) was incubated with B cells for 30 min on ice before Fc receptor blocking (422301; BioLegend). Then, the B cells were stimulated for 0, 5, 15, or 30 min at 37°C before fixation with 4% paraformaldehyde. In some experiments, B cells were stimulated for 48 h at 37°C with CpG (10 μg/mL) and CD40L (1 μg/mL) to detect activation of phospho-STAT3. The following specific antibodies were used to stain the cells after permeabilization with 0.05% saponin (Sigma): anti-CD38 (ab235118; abcam), anti-CD24 (ab202073; abcam), anti-phospho-STAT3 (9145S; Cell Signaling Technology), anti-phosphotyrosine (05-1050X; Merck Millipore), anti-pBTK (ab52192; abcam), and anti-pCD19 (3571S; Cell Signaling Technology). Images were obtained under a confocal microscope (Nikon A1R) using 405, 488, 546, 647 nm lasers. Colocalization and MFI were determined by the NIS-Elements AR 3.2 software.

PBMCs from COVID-19 recovered patients and healthy donors were incubated in a complete medium with or without 10 mM NAC (A7250-50G, Sigma-Aldrich) in 5% CO₂ for 24 h. Cells were collected and stained with PE-anti-CD19 (302208, Biolegend), then cells were incubated with 10 μg/ml biotin-F(ab′)₂ anti-human Ig(M + G) on ice for 30 min, plus 20 μg/ml streptavidin on ice for 10 min, and then activated at 37 °C for 5 min. After being fixed with 4% paraformaldehyde, cells were permeabilized with 0.05% saponin (S4521-10G, Sigma-Aldrich), and stained with anti-pBtk (ab52192, Abcam, USA), following by Alexa Fluor 647 goat anti-rabbit IgG (A-21245, ThermoFisher, USA). Samples were analyzed by flow cytometer.