Hello everyone, I'm struggling to solve this FACS staining problem:
I obtained splenocytes from a naive mouse and stimulated them with PMA+iono for 5 hours, in the presence (or not) of Brefeldin A the last 4 hours. In parallel, some wells were not stimulated.
Then I fixed with PFA 1% and permeabilized with saponin 0.1% and performed intracellular staining for IFN-g and TNF-a (since I learned these 2 cytokines should be produceed after stimulation with PMA).
BUT here are my dotplots:
1. PMA + brefeldin + permeabilization,
2. PMA without brefeldin, permeabilized,
3. PMA + brefeldin, not permeabilized,
4. not stimulated.
Why am I getting high staining in plots 3 and 4??? IMPORTANTLY, I had isotype controls with exactly the same isotype and concentrations, so this should not be unspecific binding.
I am puzzled and a little bit angry. Any help will be appreciated!
Thanks a lot!
From this.. it seems like a gating/compensation (technical) issue.
What cells are you gating on... CD3? What did the plots look like for CD3+IFNg+?
Also... 5 hours seems very short for stimulation...
You need to share your panel, and your plotting/gating strategy.
Hi,
Like Shen said , it is total compensation issue. before, you set up any experiment you need to have isotypes for every antibody.
Better is to run those compensations for 3..4 times before you go for your final acquisition.
the gating above is not correct and may need some technical guidance from some person who works their in your vicinity.
you can share your panel design and what population of cells you are looking for. May be then I can give some more insights about it.
Best,
Hamid
From a quick look at this, it appears your staining probably didn't work and you have background staining issues. There could be compensation and gain setting issues but your control seems to be the same. Why don't you first try immunofluorescence or western blot to see if you are really getting an induction before doing this experiment? With the addition of Bref A the secretory proteins will be trapped between ER and Golgi because the Golgi is shattered. All the best.
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