So helpful! Editing the matrix has always been my go-to to fix a bad compensation. 🤐

Thank you soo much .. :)

Hi Dr, Rieger. First, thanks for your channel, it has clarified a lot of questions I had. I am wondering if the gate adjustment you showed (min ~8) can be done when using unstained control (?). Digging on my data, I checked the comp area and I had to adjust the axis to enlarge the peak and only then I was able to see 2 peaks (I assumed, the positive and negative peaks). Is that expected when using unstained control for compensation? In your video boths peaks are pretty clear from the very beggining, but I used unstained control and I had to adjust the axis and then adjust the gating. Is that correct? I would appreciate your help!

Well said, lot of good points. I will suggest users to bring the negative population from a completely unstaned bead or cell. The negative bead or cell in the single stain will have some level of NSB that will lead to violation of rule of AF. DIVA only allow one universal negative but using more than one universal negative will be a good idea (cells for LD stain and Beads for Ab stain) for analysis.

Thank you very much for your videos. I have rather a simple question and would greatly appreciate any advice. What do you use for your dead cell exclusion? I am confused because I use 7-AAD and it spills over to my other channels of TMRM and MitoSOX Red dyes, hence I can't exclude dead cells prior the compensation matrix formation. I can't use other viability dyes because they overlap even worse with my other dyes in the panel of 5 dyes.

What if the cell population cannot be separated well within a single color control for compensation? How to identify negative and positive peak in this single color for compensation? Thanks!

I made the mistake of reusing my compensation matrix because I have a very large panel (18 colors). My matrix only had one major spillover issue of 153% but the outcome is that it is showing my TCRb+ T cells expressing IgG's :') everything else looks fine, its even been looked at by Flowjo people (briefly) :( I can't redo it either as I did it on brain cells and have nothing banked (near impossible to bank brain cells after stroke). I am trying to graduate in the spring and I am STRESSED.