Anti-mTIM-1 Ab(Clone:3B3) was used in flow cytometry to detect TIM-1 expression on T cells. When I use the same Ab to repeat this experiment, I always got the negative results. I think Staining procedure is fine because I have positive control(Renca cells transfacted with TIM-1). So what is the problem?
it Is always much easier to detect transfected cells in which the target is overexpressed. you can do a titra of transfection to see what is the low detection limit. If the endogenous level is low, it is hard to get a good signal. Think whether you can stimulate to upregulate your target. Also try another antibody like a polyclonal Ab in case your current Ab activity is low.
Using a directly conjugated primary antibody to PE or an equivalently bright fluor is usually recommended for low expression markers. Titration is quite important, some antibodies need 2ug per 1million cells in 100uL staining volume. It is often less for PE conjugates but, using that as your top end, titrating by 1/2 down to 0.06ug per stain should provide you an effective range. Using a biotin primary +streptavidin-PE conjugated secondary can also help provide some signal amplification however both the primary and streptavin should be appropriately titered to avoid background. hope that helps!
I already used Renca cells transfected with TIM-1 as positive control. In this case, I used 1 μL primary and 1μL PE-conjugated secondary Abs, which did produce significant signal. So do I still need Ab titration for T cells?
Hi Zhengzuo, I think so yes additional titration will be telling. 1uL of purified antibody is usually between 0.2 and 1 ug of antibody depending on your antibody concentration (if known). When we tested mTIM1 clone 3B3 we found the optimal staining concentration at 0.75ug per million cells in 100uL staining volume on cell line, however, we also do not see TIM1 staining on resting splenocytes from B6 mice. You may need to look at stimulation conditions to upregulate expression, took a look at the lit, but nothing obvious popped up. Good luck!
@Avi Perna. Thank you so much. So did you finally see TIM-1 expression on spleen cells? What do you add to upregulate TIM-1 expression?
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