Fitc anti iga

HEK293T cells were transfected with a plasmid-expressing wild-type SARS-CoV-2 S protein^{53 (link)}. Next day, transfected cells were collected and incubated with sera or recombinant anti-SARS-CoV-2 Spike Glycoprotein S1 antibody (CR3022, Abcam, Cat. No. ab273073, 1 µg/mL) for 30 min, washed twice with PBS/BSA and stained with anti-human IgG-Alexa647 (polyclonal, Southern Biotech Cat. No. 2014-31, 1:400), PE anti-IgM (CH2, Invitrogen Cat. No. MA1-10381, 1:200) and FITC anti-IgA (polyclonal, Southern Biotech Cat. No. 2052-02, 1:200), anti-human IgA1 Alexa488 (B3506B4, Southern Biotech Cat. No. 9130-30, 1:200), anti-human IgA2-Alexa647 (A9604D2, Southern Biotech Cat. No. 9140-31, 1:200) for 30 min. Cells were washed with PBS/BSA and DAPI was added before analysis for dead cell exclusion. Samples were analyzed on a FACSCanto (BD Biosciences) and using FlowJo v10 (Tree Star Inc.) analysis software. Mean fluorescent intensities were determined for transfected and non-transfected cells of each sample. Data were presented as ΔMFI = MFI(transfected)−MFI(non-transfected).

Mouse primary splenocyte suspension was collected and stained with combinations of the following antibodies: BV421 anti-IgG, AF750 anti-CD19 (Biolegend), PE anti-CD138 (Syndecan-1, R&D Systems), and FITC anti-IgA (Southern Biotech). For intracellular detection of IgG and IgA, Golgi-stop was added to cell cultures 4 h prior to harvest and extracellular staining was performed using anti-CD19 and anti-CD138 Abs. Cells were then fixed and permeabilized before intracellular staining with anti-IgG and anti-IgA Abs.Stained cells were analyzed on an Attune NxT flow cytometer (Thermo Fisher Scientific, Waltham, MA).