Hi Taylor,

What buffer you are using for permeabilization, blocking, washing and fixing? We usually use PBS to prepare these buffer not directly water... If you need more information for buffers let me know.

Good Luck,

Hi

I have been using ebioscience Foxp3 transcription kit for last 2 and a half year and I don't find any problem regarding staining or fixation etc.

Just to be more precise a lot of factors denote your outcome. The preparation of samples, a constant 4 degree environment for cells, clear isolation from light after fixing cells and handling of samples.

The incubation period also matters for fixing and for staining of cells.

Hope my reply can add help to your queries.

Good luck.

Hi,

I have used eBioscience Foxp3 kit for 4-5 years without any problem.

I used to stain cultured CD4 T cells for various intracellular and intranuclear markers (like Foxp3, T-bet, IFNg, IL-17, IL-10, CTLA4, KI67 etc). Fixation used to be done on ice for minimum 2 hours to maximum overnight.

Make sure that before you start staining with intracellular antibodies all the traces of PFA (from the fixation step) are removed. Also please check the PH of your kit buffers.

After fixation and permeabilisation do you observe any change in the FSC-A profile of the cells on flow? (fixation and permeabilisation induce shrinking of the cells. so it is expected to see a shift in the forward scatter)

Also, after fixation do you observe any change in the cell pellet morphology? does pellet turn hard white to translucent?

Do you stain your fixed and permeabilised cells for intracellular markers in presence of permeabilisation buffer?

Hope this will help you.

All the best

Neeraja

Hi Rosemary,

Are you sure it is the kit that is the problem? Just making sure that you did add secretion inhibitors to your culture (4 hours before terminating).

FoxP3 is always hard to pick up. I recently tested 6 different clones from 2 different vendors and the best one still gave

Hi all,

Finally managed to figure it out. Seemed like a couple of factors: 1) the 647/APC channel on our flow isn't great, which explains why some of our antibodies weren't coming up (e.g. ki67 and Foxp3); 2) the permeabilisation buffer needs to be at room temp by the time it is added to the cells, we were previously keeping it in the fridge.

Thank you all for your help!

I'm currently having this problem. I know it's not the colour of the Foxp3 - neither AF700 nor FITC nor PE seems to work and our FACS machines are very good. Thank you for the tip-off with the room-temp perm buffer, I'll give that a go next. Not sure why it should matter, but sometimes that's science for you. The last resort is to fix/perm overnight as has been suggested on other questions...

Room-temperature perm/fix buffer did the trick!!! Thank you so much Rosemary! I tried 4°C for one hour, 4°C overnight and there was absolutely nothing, even at 1/50 dilution (annoyingly cytokines stained fine, it was clearly just the nuclear permeabilisation).

The crucial steps were

- Let perm/fix buffer come to RT overnight

- Wash cells into the perm/fix buffer to remove traces of fixative

- Permeabilise in perm/fix buffer at RT for 1 hour

- Stain for Foxp3 at 1/50 at RT for 1 hour

You still have to acquire a lot of cells to see a good population, but in 1 million cells total I get a solid 2-3% of Foxp3+ like this. So relieved!

Sophie Sagawe Hi! I was wondering what kind of cells are you using? Are you using the mouse splenocytes? I did not use the kit but I used the Foxp3 antibody from eBiosciences and the normal Fixation kit from BD.

Jing Zhu I tried mouse bronchoalveolar lavage cells (the mice were infected so there were more cells in it), lung cells, lymph nodes, and spleen. The Foxp3+ are still a small population but in splenocytes you should definitely be able to see them. The normal fixation kit might not be strong enough because you need to permeabilise the nucleus too to stain for Foxp3.

Sophie Sagawe Thank you for the answer! I did try Foxp3 staining again for mouse splenocytes today. I only saw around 2-3% Foxp3+ when I gated CD4 vs Foxp3 in CD3+ population. I was wondering whether that is a reasonable number? Because Foxp3+ cells are not a distinct population, I am not 100% sure whether I gated the right ones (I do have FMO as reference though). Also, can I know whether you use live/dead dye when fixing cells? I saw some Foxp3 staining in non-CD4 cells which should be non-specific staining. I did do Fc block so I am guessing adding the live/dead dye can help remove the non-specific staining?

Jing Zhu I think maybe in spleen you would expect a few more (up to 8% maybe?), but with the FMO you should be able to gate accurately. Live/Dead staining is an absolute must, in addition to Fc block.

Sophie Sagawe Okay. I will add Live/Dead dye next time. Thank you!!!

This is what I do. I tested fix/perm times to save GFP expression (Foxp3GFP mouse) to able to check cytokines produced by Tregs. It works consistently well regardless of the machine (LSRII, Canto, Cytoflex). Staining is done on 96 well, round bottom plate (Corning #3797) with 5-10ul of antibody cocktail. Foxp3, Ki67 and cytokines work well using Foxp3 buffer set (eBioscience). After permeabilization, cells need to be kept in Perm buffer, antibodies need to be prepared in Perm buffer. It should be 5-10% Tregs in LN, spleen and 15-25% in colon. Or just use Foxp3 reporter mouse :-)

Hi,

I have been using eBioscience Foxp3 transcription buffer for last 7 years and never had any problem. But you need to be careful for its storage, preparation of buffer for experiment.

You need to look after what sort of cells you are using during experiment too.If you are facing continuous problem, shift to other company's reagent to see whether that works or not.

Best,

Hamid

did anyone ever use this kit on neuronal/glial cells?

Magdalena Kurtyka