Hello Alaleh, You don't indicate what you are trying to evaluate. Here are my protocols for intracellular(cytokine) and intranuclear(histone) which I hope may assist you. The first is less aggressive, as you only need to permeabilise the outer cell membrane, the histone staining is more harsh but is needed to get into the nucleus. Best wishes

Hello Alaleh, You don't indicate what you are trying to evaluate. Here are my protocols for intracellular(cytokine) and intranuclear(histone) which I hope may assist you. The first is less aggressive, as you only need to permeabilise the outer cell membrane, the histone staining is more harsh but is needed to get into the nucleus. Best wishes

Hi Alaleh,

I have been using BD kit for intracellular staining for 5 years and it was working fine. I have been staining Th1 and Th2 cells for IFNg and IL-4. Stain cells according to manufacturer instrusctions: 10 Live/dead staining for 30 min on 4C, wash once with 2 ml of PBS, spoin down 1600rpm for 10 min, cell surface staining for 30 min, repeat wash, fix/permeabilize cells for 60 minutes, wash twice, intracellular staining for 60 minutes. That should be quite good approach. Staining and washing was done in FACS tubes. I have use 2ml for washing instead of 1ml and got improved cell count, meaning I was loosing less cells.

Hope this will help.

Milan

Dear Martin and Milan,

Thank you for your excellent advice.I read them carefully and have some questions.

Dear Martin I try to detect IL-17 and FoxP3 respectively for Th17 and Treg after my treatments, so it seems the first protocol could be appropriate for IL-17 and the next for Foxp3.Did you tried the first (for cytokine) intracellular staining protocol for both cytoplasmic and nucleus markers before ? Are not its agents effective for nucleus membrane like cytoplasmic membrane ?

If I am not mistaking in step 15 of intracellular staining protocol (for cytokine) you "resuspend cells in 100 ul containing intracellular antibody in perm buffer (FACS buffer containing 0.25% saponin)".

Does not perm buffer make change the structure (epitops) of antibodies during the incubation time?

And thanks for great tips inside the protocols. 

Dear Milan could you please let me know how many cells you start with?

I used to work with live/dead dye (absolute formaldehyde included in the process).  Not only the cell count was significantly reduced but also the FSC/SSC pattern was changed.

Really thank for your tip about using 2 ml instead of 1ml of PBS.

And the last question is about blocking. you didn't mention to block Fcs neither in surface nor in intracellular staining through the protocols. Don't you need to block them?

Thanks again,

Is it also possible to stain cells with both surface and intracellular antibodies in one step after fixation and permeabilization ? I think it makes the staining procedure shorter and may save more cells ?

Dear Alaleh,

I was starting with 2M cells, but you can also start with 1M cells. Live/dead staining I was using was purchased from BioLegend I think and I used 1;1000 dilution for 30 min on 4C. After all staining, cell pattern was quite OK.

I forgot to mention blocking. In general, blocking is not a must. I was using 1microG of Fc block/2M cells for 20 min on 4C. That was all blocking i was using. Also, you use your wash buffer which contains BSA or FBS so that also counts as a blocking.

In terms of negative controls, it is the best to use FMO approach for target molecule your're staining for.

I hope this helps,

Cheerz,

Milan

I wouldn't do that. I would first perform surface staining, wash cells, fix and permabilize and than perform intracellular staining. Be careful when you're discarding supernatant.

Milan

Thank you for your all great recommendations Milan.

Best wishes,

Alaleh

No prob and good luck with your research.

Milan

Hello Alaleh, I've tried the saponin protocol for histone staining without much success but good cell recovery. The labelling with intracellular antibodies and the washing is performed in perm solution. I use isotype/FMOs to confirm the efficiency of the washing steps. I believe the best commercial reagents we have found for FoxP3 /transcription factor staining is the kit from eBioscience. It should also work for your IL-17 labelling. The major issue we have is that this FoxP3 detection, usually quenches reporter protein fluorescence. The FoxP3 was detected by gfp-reporter expression or was replaced with anti-CD127 staining for live cell sorting. If combining surface and intracellular, I would perform your surface staining before fix/perm for intracellular components. Try and avoid tanden conjugates and stick to robust fixable, or non overlapping fluorophores, if you have enough lasers and filters in your cytometer.

Best wishes

Thanks for your wonderful help dear Martin. Your recommendations were very enlightening.

Best,

Alaleh