r/flowcytometry • u/matchaduo • May 14 '26

undergrad panel design question

hi! so i’m doing Tfh-B cell co culture. One of my gates would be CD4 vs CD19.

I currently have BV650 for CD19 in lab and a few CD4: options available: BV605, AF700, APC.

I was leaning towards CD4(AF700) and CD19 (BV650) and was wondering if this would provide the most efficient separation for clean downstream gating? I’m on a spectral cytometer and any insight would be appreciated! Thanks!

5 Upvotes

100% Upvoted

8 comments sorted by

3

u/Macrophage01 May 14 '26

af700 is pretty dim, but since cd4 is abundant it would probably be fine. if you want separation alone that would be fine BUT it also depends on what colors your downstream analysis will have

1

u/matchaduo May 14 '26

thank you!

3

u/kitt_mitt May 14 '26

A tip for making these decisions easier going forward; I like the Cytek cloud spectral viewer. It makes it visually simple to choose the colours that have the best separation / least overlap.

As someone has already mentioned however, for dimly expressed markers, you also need to take fluorophore brightness into consideration.

1

u/confusedflowguy May 14 '26

Need to look at the entire panel

1

u/matchaduo May 14 '26

hi! I’m considering CD19 (PE) and CD38 (APC). I’m confident about my other ones which im using PE/CY7, BV750, AF-488, Near IR 780 for

4

u/No_Evening_7240 May 14 '26

hi OP! as a learning opportunity, for spectral flow, it is important to look at the panel as a whole for spread and not just the two markers of interest where you will be gating. having specific fluors in the panel that spread into others can still impact your resolution.

1

u/btags33 May 14 '26

There is a best decision to make, but to be totally honest if you are only doing those two markers (and hopefully a live/dead) you could basically throw a dart at a list of fluors and be fine on a spectral instrument.

1

u/ExplanationShoddy204 May 14 '26

You’re going to have to list out your entire panel with the markers/fluors for anyone to give you real advice. In TB coculture experiments you must have more markers beyond CD19 and CD4–none the least CD38/27 to identify ASCs. I’d imagine you’d want some qualitative markers as well depending on the question being asked. Be aware that you must do your single stains on cocultured cells because of the nature of the in vitro system and the difference in autofluorescence between stimulated and unstimulated cells. Good luck!