The conjugated antibodies for FACS were purchased from BioLegend except for anti-T-Bet (clone 4B10) and RORγ (clone B2D), which were purchased from eBioscience. To recover cells from the pancreas, the whole organ was minced and treated with colagenase P (Roche) followed by mechanic disruption and filtering through a cell strainer to obtain a single cell suspension. Lymphocytes were then harvested by gradient centrifugation over Histopique-1077 (Sigma). For intracellular cytokine staining, cells were restimulated with PMA (50ng/ml) and ionomycin (1µg/ml) with GolgiPlug™ (BD Biosciences) for 4 hours. Cells were stained for surface markers, fixed and permeabilized, and finally stained with anti-cytokine antibodies. In some experiments, anti-T-Bet and/or anti-RORγ were included to stain these transcription factors. FACS data were collected on FACS Calibur or LSR-Fortessa (BD). FACS data were analyzed using FlowJo software (Tree Star).
Anti t bet clone 4b10
Manufactured by Thermo Fisher Scientific
Anti-T-Bet (clone 4B10) is a monoclonal antibody that binds to the T-Bet (TBX21) transcription factor. T-Bet is a key regulator of T-helper 1 (Th1) cell differentiation. This antibody can be used for the detection and quantification of T-Bet expression in various cell types and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Golgiplug, by BD (1 mentions)
Facscalibur, by BD (1 mentions)
Lsrfortessa, by BD (1 mentions)
Facscanto, by BD (1 mentions)
Efluor 780, by Thermo Fisher Scientific (1 mentions)
Efluor 506, by Thermo Fisher Scientific (1 mentions)
Fitc conjugated anti cd4 mab clone gk1.5, by Thermo Fisher Scientific (1 mentions)
2 protocols using anti t bet clone 4b10
1
Isolation and Analysis of Pancreatic Lymphocytes
2
Immunophenotyping of Splenic T Cells
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Single cell suspensions of splenocytes, prepared as described above, were adjusted to 1–2 × 106 cells/ml and stained with viability dye (eFluor® 780 or eFluor® 506; eBioscience, San Diego, CA). Prior to immunophenotyping, Fc receptors were blocked with anti-mouse CD16/CD32 monoclonal antibody (mAb) (clone 2.4G2; BD Biosciences, San Jose, CA) and the cells were surface stained with FITC-conjugated anti-CD4 mAb (clone GK1.5; eBioscience) and PE-conjugated anti-CXCR3 mAb (clone CXCR3-173; eBioscience). For intracellular staining, the cells were surface stained, fixed, and permeabilised using an intracellular fixation kit (eBioscience) according to the manufacturer's instructions, and intracellularly stained with APC-labeled anti-Foxp3 (clone FJK-16s; eBioscience) and anti-T-bet (clone 4B10; eBioscience) mAb. For each antibody, staining was compared to cells stained with an appropriate isotype control antibody. Flow cytometry was performed using a FACSCanto (BD Biosciences) for acquisition, and data were analyzed using FlowJo software (TreeStar).
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