Detection of perforin, GZA and GZB was performed on mixed cell populations after dead cell removal as described [10 (link), 11 (link)]. Cells were surface stained first and then fixed and permeabilized with Cytofix/cytoperm kit (BD Bioscience, Franklin Lakes, NJ) according to instructions. Intracellular staining of perforin, Granzyme A and B were done using combinations of the following antibodies: anti-human Perforin-PE/Dazzle, Granzyme A-AF647, Granzyme A-PerCp-Cy5.5, Granzyme B-AF647 (BioLegend, San Diego, CA) and Granzyme B-BV421 (BD Bioscience, Franklin Lakes, NJ) as described [10 (link), 11 (link)]. Analysis was performed on BioRad ZE5 flow cytometers (BioRad) using Everest software and data analyzed with FlowJo software (Tree Star, Inc. Ashland, OR). Expression of surface markers was measured by the percentage of positive cells. Fluorescence minus one (FMO) strategy was used to establish appropriate gates.
Granzyme b af647
Manufactured by BioLegend
Granzyme B-AF647 is a fluorescently labeled antibody that specifically binds to granzyme B, a serine protease found in the cytotoxic granules of natural killer cells and cytotoxic T lymphocytes. This product can be used to detect and quantify granzyme B-expressing cells by flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Granzyme a af647, by BioLegend (3 mentions)
Granzyme a percp cy5, by BioLegend (3 mentions)
Granzyme b bv421, by BD (3 mentions)
Cytofix cytoperm kit, by BD (3 mentions)
Biorad ze5 flow cytometers, by Bio-Rad (1 mentions)
Cd39 pe, by BioLegend (1 mentions)
Tim 3 fitc, by BioLegend (1 mentions)
Ifn γ fitc, by BioLegend (1 mentions)
Tnf α apc, by BioLegend (1 mentions)
Il 2 pe, by BioLegend (1 mentions)
Lsrfortessa, by BD (1 mentions)
4 protocols using granzyme b af647
1
Intracellular Cytotoxic Molecule Analysis
2
Multiparametric Immune Cell Profiling
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Detection of perforin, GZA and GZB was performed on mixed cell populations after dead cell removal or after stimulation of cells in the degranulation assay. Cells were surface stained first and then fixed and permeabilized with Cytofix/cytoperm kit (BD) according to instructions. Intracellular staining of perforin, Granzyme A and B were done using combinations of the following antibodies: anti-human Perforin-PE/Dazzle, Granzyme A-AF647, Granzyme A-PerCp-Cy5.5, Granzyme B-AF647 (Biolegend) and Granzyme B-BV421 (BD Bioscience).
3
Quantification of Cytotoxic T-cell Markers
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Detection of perforin (PRF), granzyme A (GZA) and B (GZB) was performed on mixed cell populations after dead cell removal. Cells were surface stained to identify CD103- and CD103+ CD8+ T cells and then fixed and permeabilized with Cytofix/cytoperm kit (BD) according to manufacturer’s instructions. Intracellular staining of perforin, GZA and GZB were performed using combinations of the following antibodies: anti-human Perforin-PE/Dazzle, Granzyme A-AF647, Granzyme A-PerCp-Cy5.5, Granzyme B-AF647 (all BioLegend) and Granzyme B-BV421 (BD Bioscience).
4
Flow Cytometry Analysis of T Cell Phenotype
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For surface staining, in vitro cultured human T cells or NCG mice-retrieved PBMCs were stained for 30 min at 4 °C with the following antibodies at 1:200 dilution: CD8a PE/Cyanine7 (BioLegend, 344711), PD-1 PB (BioLegend, 329915), TIM-3 FITC (BioLegend, 345021), and CD39 PE (BioLegend, 328207). Intracellular staining was performed as described above and the flowing antibodies were used at 1:200 dilution: TNFα APC (BioLegend, 502913), IFNγ FITC (BioLegend, 502505), IL-2 PE (BioLegend, 500306), Ki-67 PerCP/Cyanine5.5 (BD Pharmingen, 561284), and Granzyme-B AF647 (BioLegend, 515405). Samples were run on a BD LSRFortessa and data were analyzed using FlowJo (10.4).
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