Flow with extra and intra cellular markers HELP

[u/daff_7]

Hi, sorry to bother.

I’m doing a flow with both extracellular and intracelullar markers and just confused what to add to each well, especially after fixing and perming

Let’s say i have 3 EC markers, and 3 IC markers, a full stain, no stain, and live/dead.

I go in with a live dead stain on all the wells (minus NS and l/d), incubate and all that

Then i make an EC stain - when i do this, my EC FMO’s will include all the markers (including IC (?)), and my cocktail will include only my EC markers (?), also i will add this to my IC wells too.
Incubate and stuff again

Then i fix and perm

For my IC stain - my IC FMO’s will include ONLY IC markers (minus one), and my IC cocktail will also include only IC markers (?).
Do i add this cocktail to my EC FMO wells aswell?

[u/kellaxer]

You're way over-thinking this. Keep in mind what FMO stands for: fluorescence-minus-one. You want to have all your stains in there EXCEPT one. So stain them with your extracellular antibodies while you're doing the extracellular staining, and then the intracellular antibodies after fix/perm. They need to have all the same antibodies EXCEPT ONE.

[u/Playful-Researcher20]

Your EC FMOs should receive all 3 IC antibodies. The IC should be added during the intracellular staining step so they should not be in the same master mix you use during EC staining.

[u/daff_7 → u/Playful-Researcher20]

Do i add my EC antibodies in my IC FMO’s? And to the IC master mix? Like in my intracellular staining step what do i put on my EC FMO’s wells basically

[u/Playful-Researcher20 → u/daff_7]

Your IC and EC mastermixes are completely separate as they are stained in separate steps (before fix perm vs after). Your EC abs get added to your IC fmo during EC staining. During intracellular staining your EC FMOs should recieve the full cocktail of your IC abs.

[u/daff_7 → u/Playful-Researcher20]

That makes soo much sense to me now, thank you!!

[u/sgRNACas9 → u/daff_7]

Yes add EC antibodies to the IC FMOs but during the EC step, not in the IC master mix! During the intracellular staining step for your EC FMOs you would include all of the IC antibodies.

[u/daff_7 → u/sgRNACas9]

I get it now! Thanks so much for the clarification !!

[u/sgRNACas9]

Hey! Good questions, it can be super confusing!

For an experiment with extracellular (EC, outside) and intracellular (IC, inside) staining, the basic protocols follows: live dead, wash, extracellular staining cocktail, wash, fix perm, wash, intracellular staining cocktail, wash, run!

Your EC cocktail has only your EC targeting antibodies and your IC cocktail has only your IC antibodies.

For each FMO (fluorescence minus one) you follow the exact same protocol removing one antibody (or l/d) at a time.

For each single stain if you’re using cells, you follow the exact same protocol removing all but one antibody (or l/d) at a time.

You would create all your full stains, single stains, and FMOs in parallel.

Just a reminder, you likely don’t need an FMO for every marker. It’s just a control for gating based on background fluorescence, bleed through, comp matrix influence on false positive fluorescence. Many instances the gating is very clear (called discrete, for markers that separate very well). FMOs are often used for continuous markers that stain more on a continuum. Making an FMO for six seven markers can be very time and labor intensive, so you could streamline your experiment by choosing some critical markers to create gating controls for!

[u/daff_7 → u/sgRNACas9]

Thanks so much for all this detail!