Cultured T cells were washed in PBS/0.1% BSA and re-suspended at 1×106 cells in 50 uL Brilliant Buffer (BD Biosciences) supplemented with 4% rat serum for 15 minutes at 4C. Cells were then incubated for 30 minutes at 4°C in 100 μL of Brilliant Buffer using the following antibody fluorophore conjugates (all from BD Biosciences unless otherwise noted): CD7 BV421, CD4 BV510, CCR4 BV605 (BioLegend), CD8 BV650, CD196 BV786 (BioLegend), CD3 AF488, CD45RA PerCPCy5.5, CD183 PE, CD197 PE-CF594, CD185 PE-Cy7 (BioLegend), and CCR10 APC (R&D Systems). Full details of fluorophore conjugated antibodies can be found in supplemental table 5 . Cells were then washed twice in PBS/0.1% BSA and data acquired on a ZE5 (Yeti) cytometer (BioRad/Propel Labs). Compensation and analyses were performed on FlowJo V10 (TreeStar) using fluorescence minus one (FMO) controls. Statistical analyses were performed on GraphPad Prism 7 using 2-way ANOVA with Bonferroni post-hoc corrections.
Brilliant buffer
Manufactured by BD
Sourced in United States
Brilliant Buffer is a laboratory product designed to maintain the optimal pH and ionic conditions for various biological and chemical experiments. It provides a consistent and reliable buffering system to help ensure the stability and performance of samples and reagents during analytical procedures.
Automatically generated - may contain errors
Lab products found in correlation
Cd8 bv650, by BioLegend (2 mentions)
Ccr10 apc, by R&D Systems (2 mentions)
Cd4 bv510, by BioLegend (2 mentions)
Ccr4 bv605, by BioLegend (2 mentions)
Cd3 af488, by BioLegend (2 mentions)
Cd45ra percp cy5, by BioLegend (2 mentions)
Cd183 pe, by BioLegend (2 mentions)
Cd7 bv421, by BioLegend (2 mentions)
Cd196 bv786, by BioLegend (2 mentions)
Cd197 pe cf594, by BioLegend (2 mentions)
Cd185 pe cy7, by BioLegend (2 mentions)
Precision count beads, by BioLegend (2 mentions)
Zombie nir fixable viability marker, by BioLegend (2 mentions)
Spectroflo software, by Cytek (2 mentions)
Dnase 1, by Merck Group (2 mentions)
Collagenase type 2, by Worthington (2 mentions)
Trizol ls reagent, by Thermo Fisher Scientific (2 mentions)
Attune nxt flow cytometer, by Thermo Fisher Scientific (2 mentions)
Facsaria 2 and 3 machines, by BD (2 mentions)
Live dead fixable aqua dead cell stain, by Thermo Fisher Scientific (2 mentions)
14 protocols using brilliant buffer
1
Multiparameter Flow Cytometry of T Cells
2
Multiparameter Flow Cytometry of T Cells
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Cultured T cells were washed in PBS/0.1% BSA and re-suspended at 1×106 cells in 50 uL Brilliant Buffer (BD Biosciences) supplemented with 4% rat serum for 15 minutes at 4C. Cells were then incubated for 30 minutes at 4°C in 100 μL of Brilliant Buffer using the following antibody fluorophore conjugates (all from BD Biosciences unless otherwise noted): CD7 BV421, CD4 BV510, CCR4 BV605 (BioLegend), CD8 BV650, CD196 BV786 (BioLegend), CD3 AF488, CD45RA PerCPCy5.5, CD183 PE, CD197 PE-CF594, CD185 PE-Cy7 (BioLegend), and CCR10 APC (R&D Systems). Full details of fluorophore conjugated antibodies can be found in supplemental table 5 . Cells were then washed twice in PBS/0.1% BSA and data acquired on a ZE5 (Yeti) cytometer (BioRad/Propel Labs). Compensation and analyses were performed on FlowJo V10 (TreeStar) using fluorescence minus one (FMO) controls. Statistical analyses were performed on GraphPad Prism 7 using 2-way ANOVA with Bonferroni post-hoc corrections.
3
Comprehensive Immune Cell Profiling in BAL
4
Multiparametric Immune Profiling of PBMCs
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Absolute counts of CD45+ cells in whole blood were determined at the time of blood collection on fresh samples by flow cytometry with Precision Count Beads (BioLegend). PBMCs, prepared using ficoll separation, were analyzed using a panel of antibodies directed against the following antigens: CD8 BV421 (clone RPA-T8), CD20 Pacific Blue (clone 2H7), CD16 BV570 (clone 3G8), HLA-DR BV605 (clone L243), immunoglobulin D (IgD) SuperBright 702 (clone IA6-2), CD19 BV750 (clone HIB19), CD45 Alexa Fluor 532 (clone HI30), CD71 PE (clone CY1G4), CD38 PE-Cy7 (clone HIT2), CD14 APC (clone M5E2), CD4 Spark 685 (clone SK3), and CD3 Alexa 700 (clone UCHT1). PBMC samples of 0.5 to 2 × 106 cells were stained with a master-mix containing pretitrated concentrations of the antibodies, along with BD Brilliant Buffer (BD Biosciences) and Zombie NIR Fixable Viability Marker (BioLegend) to differentiate live and dead cells. Samples were run on a Cytek Aurora spectral flow cytometer using SpectroFlo software (Cytek) and unmixed before final analysis was completed using FlowJo software (BD Biosciences).
5
Multicolor Flow Cytometry of Macrophage Subsets
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BMDMs (2 × 106) from mice fed LFD or HFHCD (with/without liraglutide treatment) were labelled after 7 days in culture with anti-mouse monocyte and macrophage specific markers (BD Biosciences, Oxford, UK) (Table 1 ). Antibodies were combined to make a master mix using brilliant buffer (BD Biosciences, Oxford, UK) and PBS. Cells were stained with the antibody mix for 30 min at room temperature in the dark, washed and re-suspended in PBS before running on the Beckman Coulter CyAn Advanced Digital Processing (Beckman Coulter, Brea, CA, USA). Cells were also stained with a combination of all antibodies except one for fluorescence minus one controls for gating controls and amine C reactive positive and negative beads were stained with one antibody for single stained controls required for compensation. Flow cytometry standard (FCS) files were analyzed using FlowLogic software (Miltenyi Biotec Ltd., Surrey, UK).
Antibodies for flow cytometry
| Marker | Flurochrome | Isotype | Clone |
|---|---|---|---|
| CD45 | Brilliant ™ Blue (BB)515 | rat IgG2b, κ | 30-F11 |
| CD11c | Phycoerythrin–cyanine 7 (PE-Cy7) | hamster IgG1, λ1 | HL3 |
| Ly-6C | Allophycocyanin (APC) | rat IgM, κ | AL-21 |
| F4/80 | Brilliant violet™ (BV)421 | rat IgG2a, κ | T45-2342 |
| CD11b | BV510 | rat IgG2b, κ | M1/70 |
6
Flow Cytometry Phenotyping of T Cells
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Stimulated cells were collected, and the cell surface stained for CD3 (HIT3a), CD4 (RPA-T4), CD8 (SK1), CD27 (O232), CD45RA (HI100) and Zombie NIRTM viability dye (Biolegend, San Diego, CA, USA) in the presence of Brilliant Buffer (BD Biosciences, Franklin Lakes, NJ, USA). Cells were washed and fixed in Intracellular Fixation Buffer (eBioscience); permeabilised in eBioscience Permeablization Buffer; stained for intracellular IL-2 (JES6-5H4), IFN-γ (4S.B3) and TNF (Mab11, Biolegend); washed and assessed using the ACEA Novocyte 3000 flow cytometer (Agilent). Data were analysed using FlowJo Version X (BD Biosciences).
7
Flow Cytometry Profiling of Lung Immune Cells
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The same two lobes of the right lung were harvested from each A/J mouse (n = 6/group) for flow cytometry and incubated in digestion media consisting of collagenase (300 U/ml, Sigma), dispase (1 U/ml, Worthington), and DNAse (2 U/ml, Calbiochem) for 30 min at 37 °C with stirring. Cells were then passed through a 40 µm cell strainer (BD Falcon), and red blood cells eliminated with lysis solution. Single cells were resuspended in a solution of Brilliant buffer (BD Bioscience) and stained for 30 min at 4 °C with the following antibodies: CD45-Brilliant violet 510 (30F11), CD24-Brilliant violet 604 (M1/69), CD64-Brilliant violet 711 (X54-5/7.1), AI/AE- Brilliant violet 650 (M5/114.15.2), CD8-Alexafluor 700 (53–6.7), CD4-FITC (GK1.5), LyC6/LyG6-APC (rb6-8C5), CD11b-PE-Cy7 (M1/70), CD11c-PE-Cy5 (N418), CD206- Brilliant violet 421 (C068C2), CD69- Brilliant violet 785 (41.2F3), CD25-PE (3C7), CD107a-Alexafluor 647 (1D4B), and 5 μg/ml anti-mouse CD16/CD32 antibody (Biolegend) to reduce antibody binding to Fc receptors. Zombie NIR staining was used to exclude dead cells. Cells were analyzed using a Cytek Aurora equipped with 5 lasers (UV 355 nm, violet 405 nm, blue 488 nm, yellow-green 561 nm, and red 640 nm) and FlowJo x.10.0.7r2 software (Tree Star). The gating strategy used and shown in Supplemental Fig. 6 was adapted from a published protocol78 (link).
8
Detecting SARS-CoV-2-specific Memory B Cells
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To detect SARS-CoV-2-specific memory B cells, biotinylated protein antigens were individually multimerized with streptavidin (SA) fluorophore conjugates, as described here.57 Briefly, full length Spike protein (RnD Systems) was multimerized with SA-Dylight 550 or SA-Dylight 650 (Thermo Fisher Scientific) in buffer containing 50/50 mixture of 2% FBS and Brilliant Buffer (BD Bioscience) for 1 hour at 4°C. Spike protein (RnD Systems) and SA-Dy550/SA-Dy650 were mixed at a 10:1 mass ratio (∼4:1 molar ratio) freshly before every staining. Cells were first stained for 10 minutes at RT with Live/Dead Fixable Blue Stain Reagent (Life Technologies). Subsequently cells were stained with 50μl of antigen probe cocktail containing 100ng of Spike per probe (i.e., 100ng of Spike-Biotin/SA-Dylight 550 and 100ng of Spike-Biotin/SA-Dylight 650) for 1 hour at 4°C. In parallel, SA-Dylight 550 and SA-Dylight 650 probes (100ng each) not conjugated to protein were used as decoy probes to gate out non-specific streptavidin-binding B cells. Next, cells were stained with an antibody cocktail (against CD3, CD10, CD19, CD21, CD27, CD38, CD40, CD69, CD71, CD95, IgD, IgG, IgM, see Table S2 ) for 30 mins at 4°C. Finally, cells were washed and fixed with 1% formaldehyde before acquisition on a LSR-Fortessa flow cytometer (BD). Analysis of flow cytometry data was performed using FlowJo software, version 10 (BD).
9
Flow Cytometry Immunophenotyping Protocol
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Cells were stained with the antibodies listed in Extended Data Table 1&2 . Briefly, after live/dead staining with LIVEDEAD Blue (Thermo Fisher Scientific) on ice for 15 min, cells were washed and incubated with FcR blocker (Miltenyi Biotech) diluted in PBS/2% BSA on ice for 10 minutes. For surface staining, cells were incubated in an antibody mixture diluted in brilliant buffer (BD Biosciences) at 1:100 to 1:250 on ice for 30 minutes. After washing with PBS/2% BSA buffer, cells were fixed with Foxp3/Transcription factor fixation buffer (eBioscience) overnight. For intracellular staining, antibodies were diluted in Foxp3/Transcription factor permeabilization buffer at a ratio of 1:250 to 1:500, and cells were incubated at room temperature for 45 minutes. For intracellular cytokine detection, cells were stimulated using Cell Stimulation Cocktail plus protein transport inhibitor (eBioscience) in complete RPMI for 4 hours, followed by the cell staining procedures described above. Stained cells were acquired with an Aurora (Cytek Biosciences) and analyzed using FlowJo software (v10, BD Biosciences).
10
Multiparametric Immunophenotyping of Plasmablasts and B Cells
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