Flow cytometry experiments were performed as described previously41 (link). Single cell suspensions were prepared from spleen and dead cells were excluded after staining with fixable efluor 780 (Affymatrix, Santa Clare, CA) 1:1000 diluted in PBS. Cells were washed and stained with fluorophore conjugated antibodies using flow cytometry buffer containing 0.5% FBS, 2.5 mM EDTA in PBS. For T follicular helper cell (Tfh) analysis, cells were stained with antibodies against CD4 (PerCPCy5.5 conjugated clone GK1.55 from Affymatrix), PD-1 (PE conjugated clone 29F.1A12 from BioLegend, San Diego, CA), and CXCR5 (biotin conjugated clone 2G8 from BD Biosciences, San Jose, CA) were used. Biotin was detected with 1:500 streptavidin-BV421 (BioLegend). For GC B cell analysis, antibodies against B220 (BV605 conjugated clone RA3-6B2 from BioLegend), FAS (APC conjugated clone J02 from BioLegend), T/B cell activating antigen (FITC conjugated clone GL-7 from BioLegend) were used. For plasma cell analysis, antibodies against B220 (APC conjugated clone RA3-6B2 from BioLegend) and CD138 (PE conjugated clone 281–2 from BioLegend) were used. Flow cytometry data were acquired on LSRII flow cytometer (BD Biosciences) and analyzed using the FlowJo software v10 (FlowJo, Ashland, OR).
Streptavidin bv421
Manufactured by BioLegend
Sourced in China, United States, Germany
Streptavidin-BV421 is a fluorescent conjugate of the protein streptavidin and the dye BV421. Streptavidin binds strongly to the small molecule biotin, which is commonly used to label proteins, cells, and other biological entities. The BV421 dye emits fluorescence in the violet-blue region of the visible spectrum, allowing it to be detected using flow cytometry or other fluorescence-based techniques.
Automatically generated - may contain errors
Lab products found in correlation
Anti human cd19, by BioLegend (5 mentions)
Anti human cd21, by BioLegend (5 mentions)
Anti human cd27, by BioLegend (5 mentions)
Foxp3 fix perm buffer, by Thermo Fisher Scientific (4 mentions)
Anti icos, by BioLegend (4 mentions)
Anti cxcr5 biotin, by BD (4 mentions)
Anti foxp3, by Thermo Fisher Scientific (4 mentions)
Anti cd19, by BioLegend (4 mentions)
Anti cd4, by BioLegend (4 mentions)
Anti human cd3, by BioLegend (4 mentions)
Histopaque, by Merck Group (4 mentions)
Streptavidin pe, by BioLegend (3 mentions)
Cytoflex, by Beckman Coulter (3 mentions)
Anti ia, by BioLegend (3 mentions)
Anti glut1, by Abcam (3 mentions)
Anti ki67, by BD (3 mentions)
Streptavidin pe, by Thermo Fisher Scientific (3 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Lsrii flow cytometer, by BD (2 mentions)
Anti human igm, by BioLegend (2 mentions)
53 protocols using streptavidin bv421
1
Flow Cytometry Analysis of Immune Cells
2
Murine Germinal Center and Tfh Cell Analysis
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Mice were immunized subcutaneously at the tail base, and inguinal LNs were collected on day 14 post immunization. For GC analysis, cells were mechanically digested and strained through 70 µm filters prior to staining for GC B cell analysis, cells were stained for viability (Live/Dead Fixable Aqua, Thermo Fisher), CD3e (dye: BV711, clone 145‐2C11; BioLegend), B220 (dye: PE‐Cy7, clone RA3‐6B2; BioLegend), CD38 (dye: FITC, clone 90; BioLegend), and GL7 (dye: PerCP‐Cy5.5, clone GL7; BioLegend), and for antigen‐specific staining biotinylated eOD conjugated to streptavidin‐BV421 and streptavidin‐PE (BioLegend). For TFH analysis, cells were stained for viability (Live/Dead Fixable Aqua, Thermo Fisher) and against B220 (dye: BV510, clone RA3‐6B2; BioLegend), CD4 (dye: FITC, clone GK1.5; BioLegend), CD44 (dye: PE‐Cy7, clone IM7; BioLegend), PD‐1 (dye: BV421, clone RMP1‐30; BD Biosciences), and CXCR5 (dye: PE, clone 2G8; BD Biosciences). Samples were analyzed by flow cytometry on a BD Celesta and analyzed on FlowJo.
3
Generating and Staining pMHC Multimers
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pMHC monomers were generated as previously described66 (link). All pMHC-multimer reagents were generated by 45 min incubation of 4 μg biotinylated pMHC monomer with 1 μg streptavidin-BV421 (Biolegend, reference #405225) or Streptavidin-APC (eBioscience, reference #17-4317-82) in a total volume of 100 μl FACS buffer for staining of up to 1x107 cells. pMHC-multimer staining was performed separately from surface antibody staining for 45 min on ice. Surface antibody staining was performed for 25 min on ice subsequent to pMHC-multimer staining. Depending on the respective experiments, a subset of the following antibodies were used: anti-human TCR α/β FITC (BioLegend, reference #306706), CD3 PC7 (Beckman Coulter, reference #737657), CD8 PE (Invitrogen, reference #MHCD0804), anti-murine TCR β-chain APC (BioLegend, reference #109212), anti-murine TCR β-chain APC/Fire750 (BioLegend, reference #109246), CD62L FITC (Biolegend, reference #304804), CD45-RO PC7 (BioLegend, reference #304230), Lag3 FITC (Biolegend, reference #369308) and PD-1 APC (Invitrogen, reference #17-2799-42). Live/dead discrimination was performed with propidium iodide (LifeTechnologies, reference #P1304MP).
4
Identification of Antigen-Specific T Cells
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2 × 106 cells from LN cell suspensions were stained with LIVE/DEAD Fixable Blue Dead Cell kit according to manufacturer’s protocol (Invitrogen). Samples were then incubated with a titrated amount of H10-tetramer probe for 20 min at 4°C. H10-tetramer probes were prepared beforehand by mixing biotinylated H10 protein with streptavidin-BV421 (Biolegend) at a 4:1 M ratio. Samples were washed and stained with a surface antibody cocktail (Table S2 in Supplementary Material). Before intracellular staining (Table S2 in Supplementary Material), samples were fixed and permeabilized using the Transcription Factor Buffer Set (BD Biosciences). Samples were resuspended in 1% paraformaldehyde before acquisition using a Fortessa flow cytometer (BD Biosciences). Results were analyzed using FlowJo version 9.7.6.
5
Biotinylated HIV peptide-MHC tetramerization
6
Multiparameter Immunophenotyping Protocol
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The following antibodies were used for surface staining at 4°C for 30 min: anti-CD4 (Biolegend, RM4-5), anti-ICOS (Biolegend, 15F9), anti-CD19 (Biolegend, 6D5), anti-PD-1 (Biolegend, RMP1-30), anti-CXCR5 biotin (BD Biosciences, 2G8), T- and B-cell activation antigen (BD Biosciences, GL-7), CD38 (Biolegend, 90), CD138 (Biolegend, 281-2), and anti-CD95 (BD Biosciences, Jo2), Streptavidin-BV421 (Biolegend, 405225). For intracellular staining, samples were fixed with the Foxp3 Fix/Perm buffer set according to the manufacturers instructions (eBioscience). Samples were then intracellularly stained with anti-FoxP3 (eBiosciences, FJK-16S). No viability dye was included. Samples were analyzed on FACS Canto II (BD) or Cytek Aurora. Data was analyzed using FlowJo v10 (FlowJo LLC).
7
Multimerization of pMHC Monomers for TCR Analysis
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pMHC‐monomers were generated as previously described.60 All biotinylated pMHC‐monomers were multimerised by incubation of 4 µg biotinylated pMHC monomer with 1 µg streptavidin‐BV421 (BioLegend; San Diego, California) or streptavidin‐PE (BioLegend) in a total volume of 100 µL FACS buffer per 1 x 107 cells. The following antibodies were used: anti‐human TCR α/β PE (BioLegend), CD3 PC7 (BD Biosciences; San Jose, California), CD8α PE (Invitrogen, Thermo Fisher Scientific), CD8β PC5.5 (Beckman Coulter; Brea, California), CD45 PerCP (Thermo Fisher Scientific), CD45 ECD (Beckman Coulter), CD45 PC7 (eBioscience, Thermo Fisher Scientific) and anti‐mTRBC APC (Biolegend). Live/dead discrimination was performed with propidium iodide (Invitrogen).
8
SARS-CoV-2 Memory B Cell Detection
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Detection of SARS-CoV-2-specific memory B cells (MBCs) by flow cytometry was as previously described.21 (link) In brief, for SARS-CoV-2 specific MBCs responses, biotinylated SARS-CoV-2 Spike RBD protein (40592-V08H2-B; Sino Biological, Beijing, China) was mixed with Streptavidin BV421 (405225; Biolegend, San Diego, CA, USA) at 4:1 molar ratio for 1 h at 4°C to obtain the antigen probe. According to the manufacturer’s instruction, peripheral blood mononuclear cells (PBMCs) were stained for 30 m at 4°C using antigen probe (1:33.3) and the following conjugated antibodies: antihuman CD3 (1:50) (300430; Biolegend), antihuman CD19 (1:50) (302212; Biolegend), antihuman CD21 (1:50) (354918; Biolegend), antihuman CD27 (1:50) (356406; Biolegend). After staining, cells were rewashed and resuspended in a 200ul FACS buffer. Samples were then evaluated by flow cytometry (CytoFLEX; Beckman Coulter, Brea, CA, USA) and analyzed using FlowJo (version 10.0.7r2; Treestar, Woodburn, OR, USA).
9
SARS-CoV-2 Antigen-Specific B Cell Detection
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We used a PBMC isolation kit (Solarbio, P8900, Shanghai, China) to isolate PBMCs. First, in order to detect antigen-specific SARS-CoV-2 B cells, approximately 1.0 × 106 PBMCs were treated with LIVE/Dead Zombie Aqua Dye (423102, BioLegend; Beijing, China, 1 μL/well) at 4 °C for 15 min. Biotinylated SARS-CoV-2 RBD protein was combined with Streptavidin-BV421 (405225, BioLegend) at a 4:1 molar ratio and let to sit for one hour at 4 °C to obtain the RBD probe. Fluorescence-Activated Cell Sorting (FACS) buffer (PBS + 2% FBS) was used to resuscitate PBMCs into a cell suspension, then the sample was transferred to a microplate, ensuring 100 μL per well. Then, at 4 °C, antigen probes with a concentration of 1.5 μL per well were used for staining for 30 min, followed by these conjugated antibodies: APC anti-human CD3 (300412, BioLegend; 1 μL/well), PerCP/Cyanine 5.5 anti-human CD19 (302230, BioLegend; 1 μL/well), FITC anti-human CD21 (354910, BioLegend; 1 μL/well), PE anti-human CD27 (302808, BioLegend; 1 μL/well). After being stained, the cells were washed and resuspended in 200 μL FACS buffer. The full gating strategy is illustrated in Supplementary Figure S3A .
10
Mamu A*01 Tetramer and Monomer Preparation
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Tat-TL8 (TTPESANL) Mamu A*01-APC conjugated tetramers and Gag-CM9 (CTPYDINQM) Mamu A*01 monomers were supplied by the NIH Tetramer Core Facility at Emory University, Atlanta, GA. Gag-CM9 Mamu A*01-BV421 tetramers were prepared in our laboratory using Gag-CM9 momomers and streptavidin-BV421 (Biolegend, San Diego, CA) with protease inhibitor cocktail set I (Calbiochem, Billerica, MA).
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