Yixiong, can you please post some images of your gating for your flow cytometry? It is easier to make an interpretation if I can see what you are analyzing.

This is tricky to answer without knowing your setup. Is the change in position on FSC/SSC plots or for the plots involving a colour? If the latter, then it could be due to the colours interfering with one another. That way, cells that stain positive for one marker may shift in a different colour due to a spillover. If this is the cause, compensation may fix the problem.

Well, think about what is different between two samples having different position. If the ab concentration is the same in two samples having a different position, it's probably not caused by high ab concentration. There must be something else - for example, some inhibitors cause the cells to shrink. Find any difference between the samples and start there.

In flow cytometry the term "position" can be easily misunderstood. Are we talking about a FSC SSC dot plot , afluoresence dot plot or a combination of both?

Maybe it is a good idea to post some images. Anyway, Ab concentration is a false problem, maybe your Ab actually cause a cellular response...i.e. a change of "position" of your cells. Please post some image, maybe the community can help .

Thanks!

When I say position, I mean fluoresence dot plot(not FSC/SSC)

Here is some images showing what I mean.

I tried to stain TGF-beta in treg cells and IFN-gamma in CD25- PBMC

I stimulated the cells and tried to detect the cytokines in different time point.

 Even in the same setting, you can see how obvious the position changes and it makes me hard to determine the gate.

Here is my exeperiment setting:

Stimulation before FCM: PMA(20ng for 1ml cells in 24 well plate),ionomycin(500μg for 1ml cells) GS(0.67μl for 1ml cells) for 4h

10^5 cells/FACS tube

BD Cytofix/Cytoperm™ for permeabilization

TGF-b(PE),2µl/test, BD Cat.#562339

IFN-g(PE),2µl/test BD Cat.#554701

permeabilization: 20min

ab incubation: 30min

FACS machine：BD CantoII

Hi Yixiong,

I just realised you are at Westmead too! Once Maggie is back from leave, she is bound to be able to help. Steve Schibeci also has a lot of flow experience.

All I could suggest is adding an isotype control to determine your gate since the cells may naturally fluoresce differently depending on how they respond to stimulation over time. You could then see where the gate ought to be to figure our how many cells are positive. I also note that these samples are indeed compensated. If there is uneven expression of markers in the other colours, that can lead to the cells moving about.

Dear YiXiong, two quick suggestions: 1) negative control for 12h (maybe nothing is chanfìged, maybe not...flow is often full of surprises!) 2) an isotype control for both time points.

In my experience, isotype is sometimes not reliable.

Because as my sample fluctuate or stay high in position, the isotype tend to stay in the same lower position.

For exemple(see the attached image), I used FOXP3(PE) to stain treg cells(which should be FOXP3+) as well as PE isotype. If I gate FOXP3+ according to the PE isotype, it will be almost 100% positive FOXP3! But it makes more sence to me if I only take the "smear of cells" as the positive population(showed in the image: 4.81%).

In terms of gating positive cells, now I am only confident when I see the positive and negtive population at the same time in the sample: either two seperate peaks or a "smear" of positive cells above negtive population(you can see what I mean in another question with image: https://www.researchgate.net/post/How_do_you_define_positive_and_negtive_populations_in_your_flow_cytometry_results)

To Helen Williams: Hi! Nice to meet you here.

Maggie helps a lot. I will continue ask her questions.

Good luck!

For anyone still following this:

We had a similar problem and it may be down to the FOXP3 buffer kit from ebio which is commonly used for intranuclear stains. We think it is down to incorrectly washing the cells in facs buffer without first washing with perm buffer.

We got round this by the following addition to the protocol:

Wash off the intracellular-directed antibodies using 1X perm buffer.

Then add an additional step where you wash again in normal facs buffer. 

I also think it is due to the concentration of the antibody. A simple titration experiment with different concentrations of the antibody would help solve this query.

I've heard before this problem may be related to the previous contamination of the flow cytometer and influence of remaining previous samples to yours due to the incomplete washing of the instrument.