After incubation, cells were harvested, washed, and stained for viability in PBS for 15 minutes (Live-dead Zombie Aqua dye), followed by surface staining for CD3, CD4, and CD8 (BioLegend) for 20 minutes at 4°C. Samples were then fixed and permeabilized using the intracellular staining kit, according to the manufacturer's instructions (BioLegend), and incubated with monoclonal antibodies against tumor necrosis factor (TNF)-α (clone monoclonal antibodies), interferon (IFN)-γ (clone B27), and granzyme B (Grz B) (clone GB11) (BioLegend) for 30 minutes in the dark at 4°C. Samples were acquired on a BD LSR Fortessa TM X-20 instrument (BD) and analyzed using FlowJo (FlowJo v.10.8.1). Mean fluorescence intensity from each peptide-exposed condition was normalized to the unstimulated control. The representative gating strategy is shown in eFigure 1 .
Interferon ifn γ clone b27
Manufactured by BioLegend
Interferon (IFN)-γ (clone B27) is a lab equipment product that detects the presence of the IFN-γ protein. IFN-γ is a cytokine that plays a crucial role in immune responses.
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Lab products found in correlation
2 protocols using interferon ifn γ clone b27
1
Multiparametric Flow Cytometric Analysis
2
Antibody Discovery Using Retrocyte Display
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AGEN1884 (anti-CTLA-4; IgG1), AGEN2041 (anti-CTLA-4; IgG2) and AGEN2034 (anti-PD-1; IgG4) were discovered using a proprietary mammalian display technology, Retrocyte Display™ [26 (link)]. The full sequences for each antibody are publicly available [27 , 28 ]. Notably, the variable heavy (VH) and variable light (VL) chains for AGEN1884 and AGEN2041 are identical. IgG1, IgG2 or IgG4 variants of an antibody selective for the human cytomegalovirus (CMV) protein, glycoprotein B (clone SM5-1 (4Ab-028)), for which the sequence is also publicly available, were used as isotype control antibodies [29 (link), 30 ]. The antibodies used for flow cytometry analysis of human peripheral blood mononuclear cell (PBMC) populations included: anti-CD3 (clone OKT3), CD4 (clone OKT4 or L200), CD8 (clone SK1), CD25 (clone BC96), CD28 (clone CD28.2), CTLA-4 (clone L3D10), interferon (IFN)-γ (clone B27) and tumor necrosis factor (TNF)-α (clone Mab11) purchased from BioLegend, and anti-Forkhead box P3 (FoxP3) (clone PCH101) purchased from Thermo Fisher Scientific.
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