Cells were centrifuged for 5 min and resuspended at a final concentration of 1e6 cells/100 µL in Cell Staining Buffer (BioLegend #420201); 100 µL of cells were plated in a 96-well V-bottom plate and washed with 100 µL of 1X PBS. After centrifuging the samples at 500× g for 5 min at 4°C, the supernatant was discarded and washed again with 200 µL of 1X PBS. The cells were then resuspended in diluted Live/Dead Aqua (Thermo Fisher Scientific, L34967) and incubated on ice for 30 min protected from light. Cells were washed with 150 µL of 1X PBS and centrifuged at 500× g for 5 min at 4°C. Surface antibodies were suspended in Cell Staining Buffer (BioLegend, 4420201) with Brilliant Stain Buffer plus (BD, 566385) prior to antibody addition. Cells were blocked and stained as described above in section X. Cells were then fixed using BD Cytofix/Cytoperm Buffer (BD, 554722, 554714) and incubated for 20 min at 4°C. The samples were spun at 500× g at 4°C for 5 min and the supernatant was removed. Cells were resuspended in 200 µL Cell Staining Buffer and then strained with a cell strainer cap before running on a 3-laser Aurora spectral cell analyzer (Cytek Biosciences).
Brilliant stain buffer plus
Manufactured by BD
Sourced in United States
Brilliant Stain Buffer Plus is a laboratory reagent used in the preparation of samples for flow cytometry analysis. It is designed to maintain the stability and integrity of cellular samples during staining procedures.
Automatically generated - may contain errors
Lab products found in correlation
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (7 mentions)
True stain monocyte blocker, by BioLegend (6 mentions)
Facsymphony a5, by BD (5 mentions)
Lysing solution, by BD (4 mentions)
Spectroflo software v3, by Cytek (4 mentions)
Aurora spectral flow cytometer, by Cytek (4 mentions)
Dnase 1, by Merck Group (3 mentions)
96 well u bottom plate, by Corning (3 mentions)
Paraformaldehyde (pfa), by Biotium (3 mentions)
Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (3 mentions)
Zombie nir, by BioLegend (3 mentions)
Anti cd16 32 trustain fcx, by BioLegend (3 mentions)
Ic fixation buffer, by Thermo Fisher Scientific (3 mentions)
Cellcapture software, by Stratedigm (3 mentions)
Spectroflo v3, by Cytek (3 mentions)
Spectroflo software, by Cytek (3 mentions)
Perm wash buffer, by BD (2 mentions)
Brilliant stain buffer, by BD (2 mentions)
Trustain fcx, by BioLegend (2 mentions)
Biotinylated s1 and rbd probes, by BioLegend (2 mentions)
43 protocols using brilliant stain buffer plus
1
Multicolor Flow Cytometry Staining
2
Multiparametric Flow Cytometric Analysis of T Cell Subsets
3
Quantifying SARS-CoV-2 Spike-Specific B Cells
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We measured frequencies of RBD− and NTD-specific B cells (RBD+S1+ and NTD+S1+, respectively) among total B cells by spectral flow cytometry using a 19-color subset (Table S24 ) of a previously described 21-color panel (62 (link)). Briefly, the biotinylated spike proteins were tetramerized with fluorescently labeled streptavidin at a molar ratio of 4:1 as described previously (63 (link)), with minor modifications (62 (link)). Cryopreserved PBMCs were thawed and stained with Zombie NIR Fixable Viability Dye, followed by staining with a cocktail of mAbs and four fluorochrome-conjugated spike proteins in staining buffer (2% FBS/PBS) supplemented with Brilliant Stain Buffer Plus (BD Biosciences). The stained cells were fixed (Lysing Solution, BD Biosciences) and acquired on an Aurora spectral cytometer using SpectroFlo Software v3.0.1 (Cytek Biosciences). The analysis was performed with FlowJo v10 (BD Biosciences). The CIRC0041 baseline plot is shown as an example of our gating strategy (Fig. S9 ).
4
Spectral Flow Cytometry for SARS-CoV-2 Spike-Binding B Cells
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A 21-color panel was developed to phenotype B-cell populations and identify spike-binding B cells among PBMCs by spectral flow cytometry (Table S6 ). The biotinylated spike proteins were tetramerized with fluorescently labeled streptavidin (SA) at a molar ratio of 4:1 as described previously (Kardava et al., 2022 (link)) with following modifications: S1-B.1 was conjugated with SAR-Phycoerythrin (PE), RBD-B.1 with SA-BV421, RBD-BA.1 with SA-Allophycocyanin (APC), NTD-B.1 with SA-Alexa Fluor 488, NTD-BA.1 with SA-BUV615 and S-2P-B.1 with SA-PE-Cy5.5. Cryopreserved PBMCs were thawed, and 3 × 106 cells were stained with Zombie NIR Fixable Viability Dye at room temperature for 10 minutes, followed by staining with a cocktail containing 14 monoclonal antibodies (mAbs) and 6 fluorochrome-conjugated spike proteins in staining buffer (2% FBS/PBS) supplemented with Brilliant Stain Buffer Plus (BD Biosciences) at 4°C for 30 minutes. The stained cells were fixed (Lysing Solution, BD Biosciences) and acquired on an Aurora spectral cytometer using SpectroFlo Software v3.0.1 (Cytek Biosciences) and analyzed using FlowJo v10 (BD Biosciences).
5
Barcoded Cell Immunophenotyping Protocol
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Barcoded pooled cells were resuspended in 0.6 mL of Labelling Buffer (PBS containing 5 mM EDTA, 1% BSA [weight/volume]) with 5 μg/mL TruStain FcX™ (anti-mouse CD16/32) antibody (101320, Biolegend) for 15 min at 4°C. Samples were then incubated with a backbone panel of antibodies (Table S2 Table S2
6
Comprehensive Immune Cell Profiling
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Peripherial blood mononuclear cells (PBMCs) were freshly isolated from buffy coats of anonymous human healthy donors using Ficoll-Paque Premium (GE Healthcare). Single cells were isolated from fresh liver and tumour tissues by gentleMACS Dissociators (Miltenyi Biotec) following manufacturer’s instructions. Cells were collected and washed, and counted as numbers per gram of tissue. 5∗106 single cells/tube were be collected and blocked with Mouse BD Fc Block (BD Biosciences) and True Stain Monocyte Blocker (BioLegend). For surface staining, the antibody mix was prepared with Brilliant Stain Buffer Plus (BD Biosciences) and incubated with the samples for 20 min at 4°C. For intracellular markers, the cells were subsequently permeablised with Transcription Factor Buffer Set (BD Biosciences) according to the manufacture’s protocol. Samples were stained with intracellular antibodies for 50 min at 4°C and washed by Perm wash buffer (BD Biosciences). Flow cytometry was performed using BD FACSAria Fusion or BD FACSymphony A5 Cell Analyzer (BD Biosciences). Spectral unmixing and high-parameter analysis was performed by FlowJo v10 Software using the UMAP, FlowSOM and ClusterExplorer plugins (BD Biosciences). The cell proportions and absolute number would be calculated. The antibodies used are listed in the key resources table .
7
Cryopreserved Tonsil Cell Activation Assay
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Cryopreserved tonsil cells and PBMCs were thawed quickly, resuspended in complete medium in the presence of DNase I (10 U/ml; Sigma-Aldrich), and rested at 1 × 106 to 2 × 106 cells per well in 96-well U-bottom plates (Corning) for 5 hours at 37°C. The medium was then supplemented with the relevant peptide pools (0.5 μg/ml per peptide). Negative control wells contained equivalent DMSO, and positive control wells contained SEB (0.5 μg/ml; Sigma-Aldrich). After 18 hours, cells were washed in FACS buffer and stained with anti–CCR7–APC-Cy7 (clone G043H7; BioLegend) for 10 min at 37°C. Additional surface stains were performed for 30 min at room temperature in the presence of Brilliant Stain Buffer Plus (BD Biosciences). Viable cells were identified by exclusion using a LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific). Stained cells were washed in FACS buffer, fixed in PBS containing 1% PFA (Biotium), and acquired using a FACSymphony A5 (BD Biosciences). Flow cytometry reagents are listed in table S4.
8
Comprehensive Immune Cell Profiling
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Stimulation and staining procedure as described for the AIM assay. Chemokine receptors (CCR7, CX3CR1) were stained for 10 min at 37°C followed by staining of surface markers (CD8, CD45RA, CD14, CD19, CD127, KLRG-1, CD69, CD3, CD57, CD95, CD27, 4-1BB, CD4) for 30 min at room temperature in the presence of Brilliant Stain Buffer Plus (BD Biosciences). Cells were then washed in FACS buffer and fixed/permeabilized using a FoxP3 Transcription Factor Staining Buffer Set (Thermo Fisher Scientific). Intracellular stains (granzyme B, perforin, TCF-1, granzyme K) were performed for 30 min at room temperature. Stained cells were washed in FACS buffer and acquired using a FACSymphony A5 (BD Biosciences). All antibodies are listed in Table S6 .
9
Quantifying SARS-CoV-2 Spike Protein Binding
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5 million cells per sample of PBMC, adenoid, or tonsil were resuspended in PBS with 2% FBS and 2 mM EDTA (FACS buffer). Biotinylated S1 and RBD probes (BioLegend) were crosslinked with fluorochrome-conjugated streptavidin in a molar ratio of 4:1. Fluorochrome-conjugated streptavidin was split into 5 aliquots and conjugated to Biotinylated S1 and RBD probes by mixing for 20 min/aliquot at 4°C. Cells were first stained with the viability dye, Zombie NIR (1:800 dilution, BioLegend), for 15 min at RT, washed twice and then incubated with True-Stain Monocyte Blocker (BioLegend) for 5 min. An antibody cocktail containing the rest of the surface antibodies, the fluorochrome-conjugated S1 and RBD probes, and Brilliant Stain Buffer Plus (BD) were then added directly to the cells and incubated for 30 min at RT in the dark (200uL staining volume). Cells were washed three times and fixed in 1% paraformaldehyde for 20 min at RT before washing again and collecting on a spectral flow cytometer (Aurora, Cytek). Antibodies used in this assay are shown in Supplementary Table 8 .
10
Multicolor Flow Cytometry Panel
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Cells were incubated on ice with 2 μL of anti-CD16/32 TruStain fcX (BioLegend, San Diego, CA) plus 10 μL of Brilliant Stain Buffer Plus (BD Biosciences, Franklin Lakes, NJ) for 30 min. The cells were incubated with fluorochrome-labeled mAb for 30 min at RT in the dark then were washed twice with 3 mL flow cytometry buffer (PBS + 0.5% BSA + 0.1% NaN3). Cells were post-fixed with 200 μL IC Fixation buffer (Life Technologies Corp.) for 30 min at RT in the dark, then washed again with 3 mL flow cytometry buffer, and stored at 4 °C until analysis. Flow cytometry was performed on a Stratedigm S1200Ex (Stratedigm Inc., San Jose, CA) and data were analyzed with CellCapTure software (Stratedigm).
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