ThermoFischer Scientific
T-bet Monoclonal Antibody (eBio4B10 (4B10)), eFluor™ 660, eBioscience™
50-5825-82
eBio4B10 (4B10)
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Our research aims to understand how some CD4 T cells become autoreactive in melanoma patients treated with check point inhibitors. In this experiment, we tested the expression of the typical T helper type 1 transcription factor T-BET in CD4 T cells with an anti-TBET eFluor660 antibody.
Flow Cytometry
Stimulated (PMA+Iono) healthy donor's PBMCs
Washed twice in PBS and incubated with L/D fixable aqua dye 20 min at 4°C, then washed twice in PBS.
2.4G2
Surface staining with anti-CD4 PE dazzle594, CD3 BUV395, CD45RO BUV737 etc., 30' at 4°C, in the dark, in BD Horizon™ Brilliant Stain Buffer.
Cells were fixed and permeabilized with commercial kit (FoxpP3 transcription factor kit, eBioscience) for 30 min at 4°C with fixative, 15 min at 4°C with permeabilization buffer, and stained with anti-IFN-g PE-Cy7 and anti-T-BET eFluor660 at 1/800 in permeabilization buffer.
red laser on Symphony cytometer
The antibody identifies clearly T-BET on stimulated CD4 T cells counterstained with IFN-g. Of note, the staining also works on unstimulated cells.
Gives good separation at relatively high dilution
A FMO should be done to set the positive threshold
This conjugate works best for me. Other fluorochromes are too dim or do not work.