Good day all!
I would greatly appreciate help with the following issue: Our lab is trying to characterize mouse lymphocytes for Tbet, EOMES, and PD-1. When I ran the samples the first time, the histogram for Tbet showed a single peak in the middle of the graph. I thought this was due to not blocking the Fc receptor. We have since purchased and tried using a CD16/32 BD Bioscience Fc block and it has not changed the situation at all (1 ug added for 1E6 cells in 100 ul, incubated for 30 mn at 4C). Has anyone had this issue and does anyone have any ideas as to how to fix this problem? The Tbet antibody is mouse host and mouse reactive, conjugated to PE fluorophore. Any and all help is immensely appreciated!
From what I read, I do not think Fc block is your problem. The one from BD is widely used for blocking non-specific binding through Fc receptor.
Were your voltages adjusted so that your unstained cells were really negative? Did you see clear positive/negative signals for Eomes and PD1? All the markers you are looking for are low expressed ones and most probably, what you see is just a negative peak in the middle of the graph. You could adjust it to be closer to the left by lowering your voltage for the channel that you have the antibody in (single stain control with T-bet antibody). Another thing you could do is use less of the antibody (do a titration).
Also, when trying to detect low expressed/frequency signals, it is better to not look at histograms but at cells themselves (dot plots). It is easy to miss tiny populations when looking at histograms.
Thank you so very much for the speedy reply! I did set the voltages based on the unstained cell sample and as you suspected PD-1 and EOMES did not behave too well either; although I was at least able to differentiate positive and negative peaks with those (most likely not representative of true values, however). I will definitely try titrating the antibodies using staining index. Today I tried doing so without SI calculations and realized that it would not be too beneficial as the histogram negative peak shifted roughly proportionally to the amount of antibody added for staining. Looking at the dot plots for those small populations is good advice as well that I will keep in mind for the future. Appreciate the help and am always open to more advice on improving my flow technique.
Hello Dr. Lekishvili! I did not use a viability dye. The panel we are using for the experiment is: FITC-CD8a, PE-Tbet, APC-PD1, PerCP eFlour 710-EOMES. We have a 4 chanel (FL1-4) BD FACSCalibur. The first time around I used FSC vs SSC to set a region on live cells (there was really only one large population in the plot for most of the samples, low FSC and low SSC characteristic of lymphocytes it seems) and then a second region on CD8+ (FITC) cells in combination to gate on the EOMES and Tbet plots. When I tried the titration (I went from 2 uL of Ab to 0.25 uL by serial dilution) I only stained with PE-Tbet and set a gate based on the same FCS vs SSC population (these cells were derived from mouse lymph nodes).
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