Collect data on a BD Symphony flow cytometer. Unstained samples and single-stained samples are used to set appropriate PMT voltages that have clear positive and negative populations. Compensation beads (Anti-Rat and Anti-Hamster Ig κ /Negative Control Compensation Particles Set, Cat# 552845, BD) were used to do auto-compensation. Refer to Cossarizza et al. for the guidelines for the use of flow cytometry in immunological studies (Cossarizza et al., 2019 (link)).
Symphony cytometer
Manufactured by BD
Sourced in United States
The Symphony cytometer is a flow cytometry instrument designed for high-performance cell analysis and sorting. It offers precise data acquisition and advanced fluidics technology to enable accurate and reliable cell identification and measurement.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (6 mentions)
Hepes, by Thermo Fisher Scientific (6 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (5 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (5 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (5 mentions)
Aquavivid viability marker, by Thermo Fisher Scientific (5 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (5 mentions)
Symphony flow cytometer, by BD (2 mentions)
Facsaria, by BD (2 mentions)
Cd20 bv711, by BD (2 mentions)
Igm buv737, by BD (2 mentions)
Igg bv421, by BD (2 mentions)
Cd3 bv480, by BD (2 mentions)
Cd56 bv480, by BD (2 mentions)
Cd14 bv480, by BD (2 mentions)
Cd16 bv480, by BD (2 mentions)
Cd19 bv650, by BioLegend (2 mentions)
Human trustain fcx, by BioLegend (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Fixable viability stain 510, by BD (1 mentions)
17 protocols using symphony cytometer
1
Flow Cytometry Data Collection
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2
PBMC Thawing and Multi-Parametric Flow Cytometry
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PBMC were removed from liquid nitrogen storage and immediately thawed in a 37 °C water bath. Cells were diluted dropwise into 37 °C AIM V media (Thermo Fisher Scientific #12055091) up to a final volume of 10 mL. A single wash was performed in 10 mL of PBS + BSA, pelleting cells at 400xg for 5–10 min at 4–10 °C. PBMC were resuspended 2 mL in PBS + BSA and counted using a Cellometer Spectrum. 1–2 × 106 cells were incubated with Human TruStain FcX (BioLegend #422302) and Fixable Viability Stain 510 (BD #564406) prior to staining with a 25-color cell surface panel on ice for 25 min. Cells were washed and fixed with 4% paraformaldehyde (Electron Microscopy Sciences #15713) prior to acquisition on a BD Symphony cytometer. Raw data were compensated and curated to remove unrepresentative events due to instrument fluidics variability (time gating), doublets (by FSC-H and FSC-W), and cells exhibiting membrane permeability (live/dead gating) prior to quantification using BD FlowJo software v10.6.1.
3
Immunological Markers in T1D Clinical Trial
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Complete blood counts with differentials and chemistries were measured in local laboratories. Glucose and C-peptide levels were measured in a central laboratory, the latter using the Tosoh assay. Samples were immediately cooled and centrifuged within 1 h, per protocol (Supplementary Table 2 ). Autoantibodies were measured by radio binding assay at the Barbara Davis Diabetes Center. Peripheral blood mononuclear cells were used from a subset of the participants based on availability of samples at all time points to 24 months (n = 46 abatacept and n = 48 placebo). Flow cytometry was done at the Benaroya Research Institute. Cryopreserved peripheral blood mononuclear cells were thawed and stained with a single multicolor flow cytometry panel of 31 markers (Supplementary Table 3 ) developed to define both T-cell and B-cell subsets of interest, and acquired on a BD Symphony cytometer. Compensation and analysis were performed using BD FlowJo 10 (Supplementary Fig. 1 ). The C-peptide areas under the curve (AUCs) were compared with ANCOVA, regressing on baseline level and age. The means are adjusted for age and baseline constituents using the predicted value from the fitted model substituting the average age and baseline value but expressing the treatment group effect. C-peptide was transformed using ln(AUC/120 + 1); for all other figures, the square root transformation was used.
4
Multiparameter Immune Profiling of PBMCs
5
Multiparametric Flow Cytometry Analysis
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Spleens and PLNs were homogenized using frosted glass slides and filtered after RBC lyses (where needed) to obtain a single-cell suspension. Single-cell suspensions were stained with the following Abs: anti-CD4 (RM4-5), anti-CD8 (53–6.7) anti-CD62L (MEL14), anti-CD25 (PC61), anti-CD3 (17A2), anti-CD45 (30F11), anti-CD44 (IM7), anti-GR1 (RB6-8C5), anti-CD11b (M1/70), anti-CD45.1 (A20), anti-CD45.2 (104), anti-CD19 (1D3), anti-CD45RB/B220 (RA3-6B2), (BD Pharmingen), anti-CD11c (N418), and anti-Foxp3 (FjK-16s) (eBioscience). Intranuclear Foxp3 staining was performed according to the manufacturer’s instructions. Labeled cells were analyzed using a BD Symphony cytometer (BD) and analyzed using Flowjo-10 software. Class II MHC tetramer IAg7 InsB9-23R22E MHC class II (HLVERLYLVCGEEG) and MHC class I H2-Kd InsB15-23 (LYLVCGERG) were provided by the NIH Tetramer Core (Emory University, Atlanta, GA).
6
Comprehensive Cell Immunophenotyping Protocol
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Prior to staining, cells were washed with FACS staining buffer (chilled PBS containing 1% FBS and 2 mM EDTA). If cells were used for intracellular staining, Brefeldin A at 1× (BioLegend) was added to all reagents up until the fixation/permeabilization step. Cells were stained for 15 min on ice with eBioscience Fixable Viability Dye eFluor 780 to distinguish live and dead cells and with anti-CD16/CD32 (clone 93, BioLegend) to prevent non-specific antibody binding. Cells were washed once and cell surface proteins were stained for 30 min on ice with fluorophore-conjugated antibodies. Following surface staining, cells were washed twice and analyzed directly or fixed with IC Fixation Buffer (eBioscience) for 20 min at RT for analysis the next day. For intracellular staining, cells were washed twice in wash buffer (eBioscience) and incubated with fluorophore-conjugated antibodies for at least 30 min or overnight at 4°C. Cells were washed twice with FACS staining buffer before running samples. To obtain absolute counts of cells, Precision Count Beads (BioLegend) were added to samples following manufacturer’s instructions. All antibodies used are listed in Supplementary file 1 . Flow cytometry sample acquisition was performed on BD LSRFortessa cytometer and BD Symphony cytometer, and the collected data was analyzed using FlowJo v10.5.3 software (TreeStar).
7
Measuring Yeast Protein Expression via Flow Cytometry
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Five single S. cerevisiae colonies integrated with plasmids described above were inoculated into separate wells of 96-well plates containing 150 μl of SCD-URA medium in each well and grown overnight at 30°C with shaking at 800rpm. The saturated cultures were diluted 100-fold into 150μl of fresh SCD-URA medium and grown for 5-6 hours at 30°C with shaking at 800rpm. The plates were placed on ice and analyzed using the 96-well attachment of a BD FACS Aria or Symphony cytometer. Forward scatter (FSC), side scatter (SSC), YFP fluorescence (FITC), and RFP fluorescence (PE.Texas.Red) were measured for 10,000 cells in each well. The resulting data in individual .fcs files for each well were combined into a single tab-delimited text file. YFP expression was first normalized to RFP expression per cell (henceforth referred to as YFP/RFP), then used to calculate the median value of each well. For the no-insert control, the median YFP/RFP values of all wells were averaged together. The median YFP/RFP value per replicate for all strains were then normalized to the average no-insert control value by taking the log2 difference. The average and standard error of this ratio across replicates were calculated (Fig. 2D ).
8
SARS-CoV-2 Spike-Specific T Cell Characterization
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The AIM assay (Morou et al., 2019 (link); Niessl et al., 2020a (link), 2020b (link)) was adapted for SARS-CoV-2 specific CD4 and CD8 T cells, as previously described (Tauzin et al., 2021b (link)). PBMCs were thawed and rested for 3 h in 96-well flat-bottom plates in RPMI 1640 supplemented with HEPES, penicillin and streptomycin and 10% FBS. 1.7×106 PBMCs were stimulated with a S glycoprotein peptide pool (0.5 μg/mL per peptide, corresponding to the pool of 315 overlapping peptides (15-mers) spanning the complete amino acid sequence of the Spike glycoprotein (JPT) for 15 h at 37°C and 5% CO2. CXCR3, CCR6, CXCR6 and CXCR5 antibodies were added in culture 15 min before stimulation. A DMSO-treated condition served as a negative control and Staphylococcus enterotoxin B SEB-treated condition (0.5 μg/mL) as positive control. Cells were stained for viability dye for 20 min at 4°C then surface markers (30 min, 4°C). Abs used are listed in the Table S2 . Cells were fixed using 2% paraformaldehyde for 15 min at 4°C before acquisition on Symphony cytometer (BD Biosciences). Analyses were performed using FlowJo v10.8.0 software.
9
Detection of SARS-CoV-2-specific B Cells
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To detect SARS-CoV-2-specific B cells, we conjugated recombinant RBD proteins with Alexa Fluor 488 or Alexa Fluor 594 (Thermo Fisher Scientific) according to the manufacturer’s protocol. Approximately 10 × 106 frozen PBMC from 13 convalescent donors were prepared in Falcon® 5ml-round bottom polystyrene tubes at a final concentration of 14 × 106 cells/mL in RPMI 1640 medium (GIBCO) supplemented with 10% of fetal bovine serum (Seradigm), Penicillin- Streptomycin (GIBCO) and HEPES (GIBCO). After a rest of 2h at 37°C and 5% CO2, cells were stained using Aquavivid viability marker (GIBCO) in DPBS (GIBCO) at 4°C for 20min. The detection of SARS-CoV-2-antigen specific B cells was done by adding the RBD probes to the following antibody cocktail: IgM BUV737, CD24 BUV805, IgG BV421, CD3 BV480, CD56 BV480, CD14 BV480, CD16 BV480, CD20 BV711, CD21 BV786, HLA DR BB700, CD27 APC R700 all from BD Biosciences; CD19 BV650 from Biolegend and IgA PE from Miltenyi. Staining was performed at 4°C for 30min and cells were fixed using 2% paraformaldehyde at 4°C for 15min. Stained PBMC samples were acquired on Symphony cytometer (BD Biosciences) and analyzed using FlowJo v10.7.1 (TreeStar). In each experiment, PBMC from unexposed donors (total of n = 9) were included to ensure consistent specificity of the assay.
10
Detecting SARS-CoV-2-specific B Cells
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To detect SARS-CoV-2-specific B cells, we conjugated recombinant RBD proteins with Alexa Fluor 488 or Alexa Fluor 594 (Thermo Fisher Scientific) according to the manufacturer’s protocol. 2 × 106 frozen PBMC from SARS-CoV-2 naive and previously-infected donors were prepared in Falcon® 5mL-round bottom polystyrene tubes at a final concentration of 4 × 106 cells/mL in RPMI 1640 medium (GIBCO) supplemented with 10% of fetal bovine serum (Seradigm), Penicillin- Streptomycin (GIBCO) and HEPES (GIBCO). After a rest of 2 h at 37°C and 5% CO2, cells were stained using Aquavivid viability marker (GIBCO) in DPBS (GIBCO) at 4°C for 20 min. The detection of SARS-CoV-2-antigen specific B cells was done by adding the RBD probes to the antibody cocktail listed in Table S1 . Staining was performed at 4°C for 30 min and cells were fixed using 2% paraformaldehyde at 4°C for 15 min. Stained PBMC samples were acquired on Symphony cytometer (BD Biosciences) and analyzed using FlowJo v10.8.0 software.
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