I am planning to use a dump channel, likely with a dye such as APC-Cy7 to exclude dead cells and cells not of interest e.g. CD45RA+ cells.
I am not sure whether or not I need to add this flurochrome to the compensation matrix or not, and generally, at what stage in the order of gating / controlling should I exclude the dump channel?
I imagine that if I have a decent negative and positive peak for the dump channel flurochrome, I can draw a gate for the -ve on a histogram and then apply that gate to all of my samples (essentially making it a NOT APC-Cy7 gate), and then from this gate i can put information into the compensation matrix.
I am quite confused, any help would be greatly appreciated!
Dear Byford
Even if you wish to dump APC-Cy7 you still have to perform compensation first then analyze the data .
Check spectral overlap of APC-Cy7 with the dyes in your panel using fluorescence spectrum viewer
http://www.bdbiosciences.com/in/research/multicolor/spectrum_viewer/index.jsp?_ga=1.13165232.626815263.1476894770
Thank you. Also, sorry I am using APC-H7 not Cy7.
As it is a tandem dye, if I am using APC-H7 on multiple antibodies, do I need to do a single stained compensation control for each antibody?
Use Single stained compensation of antibody with good separation between negative and positive populations.
Hi Elliot
Usually the dump channel and the viability dye are the last ones to be chosed becuase you have several dyes available that were not used in your pannel. You should have not only the single stained controls (cell or bead) but also the FMO that is very important in multicolor staining. One very good approach is to use your "dump" antibodies biotinylated so you can simply use a fluorescent Streptoavidin. In the end just gate on the negative population and proceede.
Hello and thanks for your reply - I relaise I need single stained and FMO controls for all my other colours, but are you saying that I need them for the colour to be used in the dump channel also?
Many thanks
In my opinion you should add the dump color to FMO and gate it out as you normally do,
Thanks, should I also compensate for the dump colour before gating it out?
We care about the influence of the spillover from the dump channel into any of our target channels, not the opposite. Therefore, you need to compensate the dump channel if you perform compensation using the unselected data including the positive events in the dump channel. However, you can ignore the dump channel if you do the compensation after gating out the positive events in that channel.
- Badges
- Science method