Great Antibody to Detect TIM-3 on T Cells

Results Summary

The antibody worked well to analyze expression of TIM-3 on T cells. I used this antibody on splenocytes isolated from C57BL/6 mice infected with PbA; I was able to detect a clear signal for TIM-3. The signal correlated with other co-inhibitory molecules like PD-1, LAG-3 or TIGIT. I know that recommendation from some manufacturers is to use a Polymer stain buffer (Like brilliant stain buffer from BD); I did not find this to be required than using this antibody in combination with a few other BVs, but this may depend on the individual floruorchromes/ antibodies/ manufacturers used along with this antibody. In combination with the fact that I was able to use this antibody at a concentration of 1:100 in 50 ul, I also think this antibody is fairly economical to use, which is always great. In terms of spillover, this fluorochrome also didn´t cause much trouble into neighboring channels.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 Cytek Aurora/ 5L BD LSRFortessa.