Surface markers and intracellular cytokines were detected by the following FACS anti-human antibodies: CD4 (clone RPA-T4; BD Horizon); CD45RO (clone UCHL1; BioLegend); IL-17A (clone N49-653; BD Horizon); IFNγ (clone B27; BioLegend); and Foxp3 (clone 206D; BioLegend). Prior to FACS staining, cells were incubated in complete medium with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) and 500 ng/mL ionomycin (Sigma-Aldrich) and Golgiplug (BD Biosciences) for 4 hr. To exclude dead cells, samples were stained with GhostDye Violet510 (TONBO Biosciences, CA). For intracellular cytokines and transcription factor staining, cells were fixed and permeabilized in Foxp3 fixation buffer kit (Invitrogen). pSTAT3 (clone 4/P-STAT3) and pSTAT5 (clone 47/P-STAT5) both from BD Biosciences were stained according to manufacturer’s phosflow staining protocol. Samples were collected on a BDLSRII flow cytometry, and data analysis of live CD4+CD45RO+ cells and FACS plots were generated in FlowJo (Treestar).
Ifnγ clone b27
Manufactured by BioLegend
Sourced in United States
IFNγ (clone B27) is a monoclonal antibody that specifically recognizes and binds to interferon-gamma (IFNγ), a cytokine produced by T cells and natural killer cells. This antibody can be used for the detection and quantification of IFNγ in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Cd45ro clone uchl1, by BioLegend (1 mentions)
Ghost dye violet 510, by Cytek Biosciences (1 mentions)
Golgiplug, by BD (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Foxp3 clone 206d, by BioLegend (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
Cd8 clone rpa t8, by BioLegend (1 mentions)
Tnf α clone mab11, by BioLegend (1 mentions)
Perm wash buffer, by BD (1 mentions)
Golgistop, by BD (1 mentions)
Anti human foxp3 staining set, by Thermo Fisher Scientific (1 mentions)
Il 2 clone mq1 17h12, by BioLegend (1 mentions)
Cd161 clone hp 3g10, by BioLegend (1 mentions)
Helios clone 22f6, by BioLegend (1 mentions)
Cd4 clone 13b8.2, by Beckman Coulter (1 mentions)
Facscalibur, by BD (1 mentions)
Facs moflo, by Beckman Coulter (1 mentions)
Foxp3 clone 236a e7, by Thermo Fisher Scientific (1 mentions)
3 protocols using ifnγ clone b27
1
Multiparameter FACS analysis of T-cell subsets
2
Activation and Cytokine Analysis of CAR T Cells
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The CAR T cells were suspended with pre-warmed R10 medium containing brefeldin A (20 μL/mL, Sigma-Aldrich) and GolgiStop (1.4 μL/mL, BD). The target cells were added into the wells to stimulate the CAR T cells for 6 hr in the incubator. After stimulation, the cells were stained with the viability dye followed by surface staining with CD3 (Clone OKT3), CD4 (Clone OKT4), and CD8 (Clone RPA-T8) antibodies (BioLegend, San Diego, CA, USA). After washing with PBS, the cells were permeabilized with Cytofix/Ctyoperm buffer (BD Biosciences) for 20 min at room temperature and then washed with 1 × BD Perm/Wash buffer twice. Next, the cells were stained with the intracellular antibodies, including TNF-α (Clone MAb11) and IFN-γ (Clone B27; both BioLegend).
3
Multicolor Flow Cytometry of T Cell Subsets
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For multicolor flow cytometric analysis, PBMCs were stained with the following fluorescence-conjugated monoclonal antibodies against CD4 (clone 13B8.2; Beckman Coulter Inc., Indianapolis, IN, USA), CD25 (clone M-A251; Becton Dickinson), CD49d (clone 9F10; Becton Dickinson), CD127 (clone HIL-7R-M21; Becton Dickinson), CD152 (clone L3D10; Biolegend, San Diego, CA, USA), and CD161 (clone HP-3G10; Biolegend). Cells were then fixed and permeabilized using the anti-human Foxp3 staining set (eBioscience, San Diego, SC, USA) followed by intracellular staining with monoclonal antibodies against Foxp3 (clone 236A/E7; eBioscience), IFN-γ (clone B27; Biolegend), IL-2 (clone MQ1–17H12; Biolegend), IL-17 (clone N49–653; Biolegend), and Helios (clone 22F6; Biolegend) according to the manufacturer’s instructions. The cells were analyzed on a FACS Calibur (Becton Dickinson) or FACS MoFlo (Beckman Coulter Inc.). In some experiments, the expression level of Foxp3 was evaluated using the mean fluorescent intensity (MFI).
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