Mouse splenocytes were used freshly isolated, while monkey PBMCs were stimulated after thawing and overnight resting in culture medium. Cells were plated at 1 × 106 per well in a 96-well round-bottom plate and stimulated with either SARS-CoV-2 peptide pool S-1 or S-2 or with DMSO (Sigma) as a negative control, all in the presence of anti-CD28 antibody (BD Pharmingen), for 18 h (mouse cells) or 5 h (monkey cells) in the presence of brefeldin A (Sigma). Cells were then washed to stop the stimulation, stained with Live/Dead fixable dye (Invitrogen), fixed and permeabilized with BD Cytofix/Cytoperm Kit (BD Biosciences) following the manufacturer’s instructions, and then stained with specific fluorochrome-conjugated antibodies. Sample acquisition was performed with the LSRFortessa X-20 cytofluorimeter (BD Biosciences), and sample analysis was performed with FlowJo (Tree Star). Mouse antibodies included the following: CD3 Alexa Fluor 647, CD4 BV421, CD8 BUV395, IL-17a PerCP-Cy 5.5, IFN-γ BV650, IL-2 APC-R700 (BD Biosciences), IL-4 phycoerythrin (PE), and IL-13 PE (Invitrogen); NHP antibodies included the following: CD3 AF700, CD4 PerCP-Cy 5.5, CD8 PE (BD Biosciences), and IFN-γ fluorescein isothiocyanate (FITC; U-CyTech).
Ifn γ bv650
Manufactured by BD
The IFN-γ BV650 is a fluorochrome-conjugated antibody used for the detection and quantification of interferon-gamma (IFN-γ) in flow cytometry applications. It provides a specific and reliable means to measure IFN-γ expression in various cell types.
Automatically generated - may contain errors
Lab products found in correlation
Cytofix cytoperm kit, by BD (2 mentions)
Cd8 pe, by BD (1 mentions)
Anti cd28 antibody, by BD (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
Cd4 bv421, by BD (1 mentions)
Live dead fixable dye, by Thermo Fisher Scientific (1 mentions)
Cd4 percp cy5, by BD (1 mentions)
Cd8 buv395, by BD (1 mentions)
Cd3 alexa fluor 647, by BD (1 mentions)
Il 17a percp cy5, by BD (1 mentions)
Lsrfortessa x 20 cytofluorimeter, by BD (1 mentions)
Fixable viability stain 510 fvs510, by BD (1 mentions)
Il 2 apc cy7, by BD (1 mentions)
2 protocols using ifn γ bv650
1
SARS-CoV-2 Peptide-Stimulated Immune Responses
2
Multicolor Flow Cytometry Profiling
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The following monoclonal antibodies were used and purchased from BD Biosciences: hamster anti-mouse α-CD3-Cyanin-5-phycoerytherin (PE-Cy5; clone 145-2C11); α-CD279- Allophycocyanin-R700 (APC-R700, clone J43); α-CD152-PE-CF594 (clone UC10-4F10-11); rat anti-mouse α-CD4-Brilliant Ultraviolet-737 (BUV737; clone RM4-5); α-CD8-BUV395 (clone 53–6.7); α-CD44-Brilliant Blue-700 (BB700; clone IM7); TNF-α-Brilliant Violet-711 (BV711; clone MP6-XT22); α-CD25- Cyanin-7-phycoerytherin (PE-Cy7; clone PC61); α-CD62L-BV605 (clone MEL-14); IL-2-APC-Cy7 (clone JES6-5H4); α-CD223-BV421 (clone C9B7W); and mouse anti-mouse H2Kb-BV786 (clone AF6-88.5); IFN-γ-BV650 (clone 4S.B3). Fixable Viability Stain 510 (FVS 510) was purchased from BD Biosciences and used to identify viable cells for FACS analysis. Surface staining was performed by incubation of Abs at a 1:100 dilution in fluorescence-activated cell sorter (FACS) buffer for 30 min on ice. To measure intracellular cytokine levels, cells were treated with the BD Biosciences Cytofix/Cytoperm kit according to the manufacturer’s instructions. Manual gating was performed on FlowJo software (TreeStar, San Francisco, CA).
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