For in vitro flow cytometry analysis, cells were harvested and incubated with Fixable Viability dye APC-eFluor 780 (1:1000 dilution, Ebioscience, San Diego, CA) for 20 min. For DC cultures, cells were then stained using directly conjugated antibodies against murine surface antigens, as follows: anti-CD40-PE, anti-CD80-FITC, anti-CD3-PE-Cy7, anti-CD11b-PE, anti-CD11c-APC, anti-CD86-Alexa Fluor 700, anti-CD8-BV605, and anti-MHCII-Percp-Cy5.5 (1:400 dilution, Biolegend, San Diego, CA) and analyzed with a BD LSR-Fortessa Flow Cytometer (Becton–Dickinson, Franklin Lakes, NJ). For OTI T cell analysis, flow cytometry panel was performed as described: anti-CD4-PE, anti-CD8-APC, anti-CD44-PErcp-Cy5.5, and anti-CD3 PE/CY7 (1:200, Biolegend). For ex vivo flow cytometry analysis, the directly conjugated antibodies against murine surface antigens are as follows: anti-CD3-Alexa Fluor 700 (1:50), anti-CD4-PE-Cy7 (1:100), anti-CD8-BV605 (1:100), anti-CD44-PE (1:200), and anti-CD62L-APC-Cy7 (1:100) for surface staining (Biolegend). For intracellular staining, cells were fixed/permeabilized stained using the Foxp3 staining kit (Ebioscience) using Foxp3-eFluor 450 (1:100, Ebioscience), granzyme B Alexa Fluor 647 (1:50, Biolegend) as described in the product specification sheet.
Granzyme b alexa fluor 647
Manufactured by BioLegend
Granzyme B Alexa Fluor 647 is a fluorescently labeled antibody that specifically binds to granzyme B, a serine protease found in cytotoxic T cells and natural killer cells. The Alexa Fluor 647 dye allows for sensitive detection of granzyme B expression using flow cytometry or other fluorescence-based methods.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd8 bv605, by BioLegend (1 mentions)
Anti mhc 2 percpcy5, by BioLegend (1 mentions)
Fixable viability dye apc efluor780, by Thermo Fisher Scientific (1 mentions)
Anti cd44 percpcy5, by BioLegend (1 mentions)
Anti cd3 alexafluor700, by BioLegend (1 mentions)
Foxp3 efluor450, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Anti cd62l apc cy7, by BioLegend (1 mentions)
Anti cd11c apc, by BioLegend (1 mentions)
Anti cd4 pe, by BioLegend (1 mentions)
Anti cd40 pe, by BioLegend (1 mentions)
Anti cd11b pe, by BioLegend (1 mentions)
Anti cd3 pe cy7, by BioLegend (1 mentions)
Anti cd4 pe cy7, by BioLegend (1 mentions)
Anti cd44 pe, by BioLegend (1 mentions)
Fitc anti cd80, by BioLegend (1 mentions)
Anti cd8 apc, by BioLegend (1 mentions)
Foxp3 staining kit, by Thermo Fisher Scientific (1 mentions)
Monensin, by BioLegend (1 mentions)
Ifn γ pe, by BD (1 mentions)
2 protocols using granzyme b alexa fluor 647
1
Comprehensive Immunophenotyping of Dendritic Cells and T Cells
2
Intracellular Cytokine Staining of Tumor Cells
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For intracellular cytokine staining, tumor samples or peripheral blood cells were collected 4 h after the last dose. Tumor samples were homogenized using a gentleMACS dissociator and then filtered through 70 µm cell strainers to prepare a single-cell suspension. Blood cells were lysed with a red blood cell lysis solution. Cells were stimulated with PMA + Ionomycin in the presence of brefeldin A (BD Biosciences; #555029) and monensin (#554724) for 4–6 h. The cells were stained with CD45-BV750 (BioLegend; #103157, clone 30-F11), CD3-APC-Cy7 (BioLegend; #100222, clone 17A2), CD4-Alexa Fluor 700 (BioLegend; #100430, clone GK1.5), and CD8a-Pacific Orange (Thermo Fisher Scientific; #MCD0830, clone 5H10) in the presence of purified rat anti-mouse CD16/CD32 and then fixed and permeabilized using the Foxp3/Transcription Factor staining buffer set. The cells were then stained with IFN-γ-PE (BD Biosciences; #554412, clone XMG1.2) or GranzymeB-Alexa Fluor 647 (BioLegend; #515406, clone GB11). Samples were analyzed using CYTEK Aurora spectral flow cytometry (CYTEK Biosciences, Fermont, CA, USA), and all data were analyzed using FlowJo software.
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