Brilliant staining buffer

Cells were washed in PBS and stained for viability using FVS510 (BD Biosciences) which was diluted 1:1,000 in PBS and 100 µL was added to each sample of cells and left in the dark at room temperature for 15 min and then washed twice in FACs buffer (PBS containing 0.5% BSA, 5 µM EDTA and 0.5% sodium azide). To reduce nonspecific binding, samples were incubated with 50 µL of polyclonal rat IgG (Sigma) (diluted 1:50 in FACs buffer) for 15 min on ice and protected from light, then washed once in FACs buffer by spinning at 400 g for 5 min and removing supernatant. Samples were then stained for flow cytometry by adding 50 µL of anti-CD44-FITC (clone IM7 at 1/200; Biolegend) in Brilliant Staining Buffer (BD Biosciences). Staining with TGMs used Alexa fluor-labeled proteins made as described above. TGMs labeled with AF488 and AF594 were used at a final concentration of 5 μg/mL. Samples that were also stained for intracellular antigens were fixed and permeabilized using Foxp3 Transcription Factor Buffer kit (Invitrogen, USA) and stained with anti-Foxp3-ef450 (clone FJK-16s, eBioscience, San Diego, California, USA, 1/100) in perm/wash buffer. Samples were then washed prior to analysis on a BD Celesta flow cytometer (BD Biosciences).

Single-cell suspensions of human granulation tissue samples were resuspended in PBS and stained with Fixable Viability Dye eFluor® 780 (Thermo Fisher Scientific, Austria) according to the manufacturer’s instructions. Cells were washed thoroughly in PBS and resuspended in Brilliant Staining Buffer (BD Biosciences, Germany) diluted 1:2 with PBS for labeling. Endothelial progenitor cells (EPCs) and adipose-derived stem cells (ASCs) were stained with monoclonal antibodies against the following surface markers: CD45-FITC (clone HI30, BioLegend, Germany), CD31-PE/Cy7 (clone WM-59, BioLegend, Germany), CD34-PE (clone 561, BioLegend, Germany), and CD90-BV510 (clone eBio5E10, Thermo Fisher Scientific, Austria). Stained cells were acquired using CytoFLEX S flow cytometer (Beckman Coulter Life Science, Germany), and flow cytometry data were analyzed using Flowjo™ analysis software (Version 10.8.1, BD Bioscience). Cell death of MQ-CM-treated ASCs was assessed by AnnexinV-FITC and 7AAD staining according to the manufacturer’s instructions.

Cryopreserved PBMC were thawed and rested for at least 6 hours in filtered R10 - RPMI+10%FBS (Sigma). Cells were counted and divided between two wells of a round bottom 96-well plate (1-3x10⁶/well) in a final volume of 200ul, purified anti-CD40 antibody (Biolegend) was included at a final concentration 1μg/ml. Cells were then stimulated with a pool of overlapping peptides from Wuhan sequence SARS-CoV-2 Spike (JPT technologies) at final concentration of 1μg/ml per peptide, or DMSO as an unstimulated control. Cells were incubated at 37°C overnight for 18 hours. Following stimulation, plates were spun for 2 min at 250g, and 150ul of supernatant removed and immediately frozen at -80°C. Cells were transferred to FACS tubes and washed with cold wash buffer (PBS+0.5% BSA+0.1% EDTA), Fc-block was added for 5 minutes (Biolegend), An antibody mastermix was made using 50μl of brilliant staining buffer (BD biosciences) per sample, to which appropriate volumes of antibody were added as shown in Supplementary Table 4. Antibody was added to the cell suspension and cells stained at 4°C for 30 minutes. Cells were then washed and fixed with 1.6% paraformaldehyde for 30 minutes at RT in the dark, washed and run on a BD Symphony A3 flow cytometer (BD Biosciences). Data was collected using BD FACS Diva 8 and analysis was carried out using FlowJo v10.7.1.

Enriched cells were resuspended in 50 µl Cell Staining Buffer (CSB) (420201, Biolegend, USA) plus 10% FcR-Block (130-059-901, Miltenyi) then incubated for 15 min at 4°C. Cells were labelled with biotinylated antibodies against TCR-γᵹ (130-113-502, Miltenyi), TCR-Vα24 (130-117-958, Miltenyi) and TCR Vα7.2 (130-110-957, Miltenyi) in a total volume of 100 µl of CSB for 15 min at 4°C. After washing in 2 ml CSB, cells were stained for 30 min at 4°C with α-biotin FITC (130-110-957, Miltenyi), αCD161 PE (339904, BioLegend), αCD25 PC5.5 (356112, BioLegend), αCD56 APC (318310, BioLegend), αCD3 APC-H7 (560176, BD Biosciences, USA) and αCD4 BV421 (344632, BioLegend) in a final volume of 100 µl/sample comprising CSB plus 50 µl Brilliant Staining Buffer (563794, BD). Cells were washed with 2 ml CSB then resuspended in 500 µl PBS prior to sorting on a FACSAria Fusion (BD).

Fluorescent-labeled antibodies CD3-Alexa Fluor 700 (BD 557943), CD8-Alexa Fluor 700 (BD 561453), CD14-PE-Cy7 (BD 561385), CD16-PE-Cy7 (BD 560716), CD19 PerCP*Cy5.5 (BD 561295), CD20 PerCP*Cy5.5 (BD 350955), IgG-PE-Cy5 (BD 551497), IgM-BV605 (BD 562977), and IgD-BV605 (BD 563313), which target cell surface markers, were used as 1:200-fold dilution in staining. Biotin-labeled MPER peptide PDT-081 (E₆₅₄KNEQELLELDKWASLWNWFDITNWLWYIK₆₈₃-biotin) was purchased from GenScript and coupled separately to streptavidin-BV421 (BD 562426) and streptavidin-APC (allophycocyanin, BD 555335). PBMCs were stained using the LIVE/DEAD Fixable Near-IR Dead Cell Kit (Life Technologies, L34957) for 30 min on ice. Cells were then labeled with antibodies cocktail along with MPER probes for 1 h in Brilliant Staining buffer (BD 563794) on ice. Cell population CD19+/CD20+, CD3−/CD8−, CD14−/CD16−, IgG+, IgD-/IgM− MPER double positive were sorted using BD FACSAria III sorter into individual wells of a 96-well plate containing lysis buffer and plates were immediately sealed and stored at −80 °C. The single B-cell sorting strategy is shown in the Supplementary Fig. 9.

Discrimination of viable cells was based on staining with the Live/Dead Fixable Near-IR Dead Cell Stain Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Following Live/Dead staining, pellets were resuspended in Brilliant Staining Buffer (BD Biosciences), treated with the FcR blocking reagent (Miltenyi Biotec), and stained with a multicolor antibody panel consisting of CD11c-FITC, CD40-BV711, CD80-BV480, CD83-BV605, CD86-AF700, PD-L1-BV421, and HLA-DR-BV786 (BD Biosciences). Samples were acquired on a FACSAria III flow cytometer (BD Biosciences) with automatic compensation based on single-stained mDC and analyzed in FlowLogic (Inivai Technologies). At least 25,000 CD11c+ events were recorded. The gating strategy was based on size/complexity (FSC/SSC), singlet discrimination (FSC-area/FSC-height), viability (APC-Cy7-negative), and CD11c+ (FITC) as illustrated in Figure 2. The median fluorescent intensity (MFI) was measured on CD11c+ cells, to exclude them possibly from ASCs that are CD11c-negative. The MFI was normalized to the intensity of the mDC phenotype of the given donor.