CD3 Antibody for T Cell Panels

Results Summary

I used this antibody in an 18 color T cell panel on a 5L BD LSRFortessa. BUV395 has both desirable and undesirable propertiers; On the positive, the fluorochrome induces practically no spillover into any other channel and subsequently, is a great choice for a lineage marker like CD3 that is expressed on all cells of interest (here: CD3). On the other side, I found that the signal wasn´t always strong enough the achieve a great separation. Accordingly, I would use the antibody if focussing on T cells (where TCRgd and/or CD4 and CD8 are probably also used to further gate on cells of interest), but not for a phenotyping panel where T cells have to be clearly separated from other cell types.Two more tipps: I achieved a higher stain index when staining intracellularThis antibody greatly benefited from titration; in my case, 1:400. This is true fr this clone regardless of fluorochrome, to reduce background signal.In the following, you will find my staining protocol in short. Incubation steps were performed at 4°C in the dark. 1.Count and adjust splenocytes to 1.5E6 cells, plate in a 96-well plate. 2.If required, stain cells with viability dye. Wash. 3. Stain with extracellular master mix for 20 min. Wash 2x. 4. Fixate/ Permeabilize with buffer of choice, depending on application. I used ThermoFisher FoxP3 fixation/permeabilization kit for 20 min. Wash with perm/ Wash 2x. 5. Perform intracellular staining for 20 min. Wash 2x. 6. Resuspend in FACS buffer and acquire as soon as possible. Here, data was acquired on a 5L BD LSRFortessa.