Apc anti igm clone mhm 88

SUP-T1 cells were harvested, counted and spike-expressing and control SUP-T1 cells were mixed in a 1:1 ratio. The cell mix was transferred into V-bottom 96-well plates at 20,000 cells per well. Cells were incubated with heat-inactivated sera diluted 1:50 in PBS for 30 minutes, washed with FACS buffer (PBS, 5% BSA, 0.05% sodium azide), and stained with FITC anti-IgG (clone HP6017, Biolegend), APC anti-IgM (clone MHM-88, Biolegend) and PE anti-IgA (clone IS11–8E10, Miltenyi Biotech) for 30 minutes (all antibodies diluted 1:200 in FACS buffer). Cells were washed with FACS buffer and fixed for 20 minutes in 1% PFA in FACS buffer. Samples were run on a Bio-Rad Ze5 analyser running Bio-Rad Everest software v2.4 and analysed using FlowJo v10.7.1 (Tree Star Inc.) analysis software. Spike-expressing and control SUP-T1 cells were gated and mean fluorescence intensity (MFI) of both populations was measured. MFI in control SUP-T1 cells was subtracted from MFI in spike-expressing SUP-T1 cells, and resulting values were divided by MFI in control SUP-T1 cells to calculate the specific increase in MFI. Values >2 were considered positive.

HEK293T cells were transfected to express the different SARS-CoV-2 spike variants. Two days after transfection, cells were trypsinised and transferred into V-bottom 96-well plates (20,000 cells/well). Cells were incubated with sera (diluted 1:50 in PBS) for 30 min, washed with FACS buffer (PBS, 5% BSA, 0.05% sodium azide), and stained with BV421 anti-IgG (clone HP6017, Biolegend), APC anti-IgM (clone MHM-88, Biolegend), and PE anti-IgA (clone IS11-8E10, Miltenyi Biotech) for 30 min (all antibodies diluted 1:200 in FACS buffer). Expression of SARS-CoV-2 spike was confirmed by staining with the D001 antibody (40590-D001, SinoBiological). Cells were washed with FACS buffer and fixed for 20 min in CellFIX buffer (BD Bioscience). Samples were run on a Ze5 analyzer (Bio-Rad) running Bio-Rad Everest software v2.4 or an LSR Fortessa with a high-throughput sampler (BD Biosciences) running BD FACSDiva software v8.0, and analyzed using FlowJo v10 (Tree Star Inc) analysis software, as previously described (Ng et al., 2020 (link)). All runs included three positive control samples, which were used for normalisation of mean fluorescence intensity (MFI) values. To this end, the MFI of the positively stained cells in each sample was expressed as a percentage of the MFI of the positive control on the same 96-well plate. The results shown are from one of one to two independent experiments.