Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood samples by density-gradient centrifugation using Ficoll-Paque (GE Healthcare) and stored in 10% DMSO and 90% fetal bovine serum (FBS) at −80°C. Cryopreserved PBMCs were thawed, washed, and incubated in 5% FBS in Hanks’ balanced salt solution (HBSS) for 30 minutes at 37°C. PBMCs (5 × 106) were then spun down and resuspended in 100 μl of 1.5% nonfat dry milk (Fisher Scientific) in HBSS with 10 μl of biotinylated nuclear extract and incubated on ice for 30 minutes. The cells were spun down, resuspended, and stained for 15 minutes on ice with a cocktail of monoclonal antibodies: fluorescein isothiocyanate–conjugated anti-IgD (clone IA6-2; BD Biosciences), phycoerythrin (PE)–conjugated anti-CD27 (clone CLB-27/1; Invitrogen), PE–Texas Red–conjugated anti-CD38 (clone HIT2; Invitrogen), and Alexa Fluor 700–conjugated anti-CD3 (clone HIT3a), Alexa Fluor 700–conjugated anti-CD14 (clone M5E2), Alexa Fluor 700–conjugated anti-CD16 (clone 3G8), PerCP–Cy5.5–conjugated anti-IgM (clone MHM-88), PE–Cy7–conjugated anti-CD10 (clone HI10a), allophycocyanin-conjugated anti-CD19 (clone HIB19), and BV421-conjugated streptavidin (all from BioLegend). For analysis, 3–5 × 105 events were collected for each sample using an LSR II flow cytometer (Becton Dickinson) and analyzed using FlowJo software (Tree Star).
Bv421 conjugated streptavidin
Manufactured by BioLegend
Sourced in Canada
BV421-conjugated streptavidin is a fluorescent-labeled reagent used in flow cytometry and other immunoassays. Streptavidin binds to biotin with high affinity, allowing it to be used to detect and quantify biotinylated molecules. The BV421 fluorescent dye is conjugated to the streptavidin, providing a specific detection signal.
Automatically generated - may contain errors
Lab products found in correlation
Ficoll paque, by GE Healthcare (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Recombinant s1 and rbd proteins, by BioLegend (1 mentions)
Rituximab, by Genentech (1 mentions)
Biotinylated protein l, by Thermo Fisher Scientific (1 mentions)
Facscanto 2, by BD (1 mentions)
Novocyte, by Agilent Technologies (1 mentions)
Formaldehyde, by Thermo Fisher Scientific (1 mentions)
Cd3 apc cy7 sp34 2, by BD (1 mentions)
Ki67 pe b56, by BD (1 mentions)
Live dead fixable blue viability dye, by Thermo Fisher Scientific (1 mentions)
Transcription factor buffer set, by BD (1 mentions)
6 protocols using bv421 conjugated streptavidin
1
PBMC Isolation and Immunophenotyping
2
Preparation of B Cell-Specific Tetramers
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B cell-specific tetramers were prepared as described55 (link). A biotinylated form of recombinant S1 and RBD proteins (BioLegend) was tetramerized by the addition of PE-conjugated or BV421-conjugated streptavidin (BioLegend) and used for B cell tetramer staining assays. Briefly, SA-PE or SA-BV21 was added in an amount equal to 1/5 of the monomer substrate amount. SA was added in 5 equal portions to the monomer and incubated each time at 4 °C for 20 min on a shaker. Protease inhibitor was added to the tetramers at a 1x final concentration (Sigma). The tetramers were filled up to 100 µl with 0.1% BSA in PBS and stored at 4 °C.
3
Multi-Phenotypic Analysis of Immune Cells
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Cellular composition was determined using a staining panel including antibodies against Vδ1, Vδ2, CD3, CD14, CD19, CD56, TCRαβ and TCRγδ. Extended phenotypic analysis incorporated antibodies against CD45, CD95, CD45RO, CD62L, CD27, CD45RA, CD57, KLRG1, NKG2D, DNAM1, CCR4, CCR5, CCR7, CCR8, CXCR3, CXCR4, CXCR6, PD‐1, TIM‐3, LAG‐3 and TIGIT. Binding properties of anti‐CD20 mAbs, clone 3H7 and rituximab (Genentech, Lot #3108505) were evaluated using human IgG1 isotype control and fluorochrome‐conjugated anti‐human Fc antibodies. Antibodies were purchased from BioLegend (San Diego, CA), BD Biosciences, Beckman Coulter (Brea, CA), Miltenyi or Jackson ImmunoResearch (West Grove, PA). Surface CAR expression was measured using an anti‐CAR idiotype antibody (Adicet Therapeutics, Menlo Park, CA) or biotinylated‐Protein L (Thermo Fisher) and BV421‐conjugated streptavidin (BioLegend). Samples were acquired on a BD FACSCanto™ II (BD Biosciences), a NovoCyte (Agilent, Santa Clara, CA) or an iQue® (Sartorius) flow cytometer. Data were analysed using FlowJo™ software (Tree Star, Ashland, OR) or iQue software (Sartorius).
4
Flow Cytometry Analysis of HIV-1 and B Cell Binding
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Primary CD4+ T cells infected with HIV-1-eGFP, primary B cells or Raji-Env cells were seeded in flow tubes at a density of 5.0 × 105 cells/tube. Cells were washed in FACS buffer and incubated with 9.25 μg/mL of scFv-10-1074-biotin or 9 μg/mL scFv-CD19-biotin at 4 °C for 1 h. After washing twice with FACS buffer, the cells were incubated with 10 μg/mL of BV421-conjugated Streptavidin (BioLegend Cat# 405226) in the dark at 4 °C for 20 min. Finally, the cells were washed twice in FACS buffer, fixed with 1% formaldehyde (Thermo Scientific Cat# 28908), and analyzed by flow cytometry (MACSQuant® Analyzer 16).
5
Biotinylated Spike Protein Conjugation
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Full-length, biotinylated spike protein was purchased from R&D Systems. Streptavidin-conjugated BV421 (Biolegend) was then added at a 6:1 molar ratio (biotinylated-protein to streptavidin-conjugate) on ice for 1 hour.
6
Analyzing Env-specific GC B Cell Responses
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