Cellfix buffer

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.

Cell surface staining was performed in ice-cold PBS containing 2% FBS and 2 mM EDTA (cytometry buffer) using appropriate fluorochrome-conjugated monoclonal antibodies and corresponding isotypes, all purchased from BD Biosciences (Table 3). A total of 1.5 to 2×10⁵ cells per experimental condition were resuspended in cytometry buffer containing 2 µg/mL of FcBlock (anti CD16/CD32) for 20 min at 4°C, then stained with 1 µg/mL of antibody or control isotypes for 30 min on ice in the dark. Next, cells were washed with cytometry buffer, and fixed in BD cell fix buffer (BD Biosciences) or in 4% paraformaldehyde solution before acquisition through a BD FACSCanto™ II Cell Analyzer (IPNC) or a CytoFLEX flow cytometer (Beckman Coulter). For cell viability analysis, cells were incubated with eBioscience™ Fixable Viability Dye eFluor 780 for 5 min on ice, washed in cytometry buffer before fixation. Positive eFluor dead cells were excluded from the analysis. At least 30,000 events were acquired for the phenotyping. Compensations were performed using UltraComp eBeads (Thermofisher Scientific) stained positively with each of the antibodies used in the experiment. Baseline photomultiplier tube voltages of the instrument were set using unstained cells, showing the background fluorescence of the experimental samples. Data were analyzed using FlowJo v10 analysis software package.

Monocyte derived macrophages (MDM) were treated with 100 U/ml IFN2α for 30 min or examined untreated. Cells were fixed using BD Cell Fix Buffer (10 min at 37°C) and then permeabilized using BD PermIII Buffer (30min at 4°C). Cells were stained with BV421-anti-STAT1 pY701 (BD Bioscience, cat: 562985, 1:50) and cell surface markers APC-CD68 (Miltenyi Biotec, cat: 160-109-462, REA613, 1:30). Flow cytometric analysis was performed on a BD LSRII flow cytometer. Results were analyzed using FlowJo v10.4.2.