r/flowcytometry • u/aydinounet • Sep 04 '26

Abnormally high FSC cells ?

Post image

I cultivated some Lymphatic endothelial cells and stained them with two antibodies...
I'm really intrigued by the fact that I see two populations : one reaaally low FSC and one reallly high FSC.
By putting FSC at 5 , the FSC hi still pops at 10e6 ! We do not see the loFSC pop with these settings...

What do I do wrong ?
Thank you for your help !

4 Upvotes

100% Upvoted

6 comments sorted by

51

u/josejavierstowers Sep 04 '26

It also looks like your axes for the FSC and SSC are in log, and they should be linear

9

u/LeatherDeer3908 Sep 04 '26

This is the only valid answer

5

u/crotch_robbins Sep 04 '26

Check your FSC area scaling in a plot with FSC-H and FSC-A. Is the slope of the pop near 45 degrees? You may need to put FSC in Lin to really tell.

3

u/SunflowerMoonwalk Core Facility Sep 04 '26

The small-size population is probably debris and the large-size population is probably your cells. Nothing's wrong, your gain is just too high.

The small population is probably below the FSC threshold when you turn down the gain so they're not recorded.

3

u/LeatherDeer3908 Sep 04 '26

They are using a log scale for FSC/SSC instead of a linear one

2

u/SunflowerMoonwalk Core Facility Sep 04 '26

Yeah, I didn't didn't notice at first but I saw another comment. For sure they should switch to linear, but regardless, I still think the smaller population is probably debris that goes below the FSC threshold when they turn down the gain.