Hi,
Here I want to analyze the T cell (from mice colon tissue), including Th1, Th17 in flow cytometry, Considering the cells number, what's the length of the colon do I need for cell collection for flow cytometry? if I only use distal colon tissue for flow cytometry, will the cells enough for T cell or macrophage analysis or detection in flow cytometry? Many thanks!
Hi Xin,
In our lab, we can get adequate cell numbers for T cell analysis from 150-300 mg of colon tissue. The number of immunocytes that you can isolate from the tissue will vary greatly depending on the level of inflammation in your model, however. If you are looking for T cells within a normal, non-inflamed colon I suggest using more tissue.
Hi Xin,
In our lab, we can get adequate cell numbers for T cell analysis from 150-300 mg of colon tissue. The number of immunocytes that you can isolate from the tissue will vary greatly depending on the level of inflammation in your model, however. If you are looking for T cells within a normal, non-inflamed colon I suggest using more tissue.
Hi Xin,
When I have done DSS-induced colitis models in the past, you can usually get around 10-25 million cells per ~300 mg, which I am guessing your tissue will be, depending on how many cycles of DSS it has gone through. Cell number can also vary a lot with different protocols as well. I can offer our protocol, if you would like.
It's hard to say what length of colon tissue you should use, as the length of the colon will shorten significantly with chronic inflammation.
Hi Valerie,
Please send the protocol to me at your convenience.
The mice will have total four cycles DSS treatment to induce chronic inflammation. I wiill quantify the Macrophage, Neutrophils, DC , T, B cells at the end of each cycle. DO you know at which cycle the T,B cells in the colon will increase? For the flow cymentry, considering the cell number in the distal colon tissue, I would prefer to stain the antibody in one tube to identify all these cell I mentioned above, do you have some suggestion for the gating strategy? Thanks.
Hi Xin,
I just sent the protocol. I am not sure at which cycle the T and B cells will increase, that is an interesting question.
For your flow cytometry, I would suggest staining the granulocytes (Macrophage, Neutrophils and DC's) and the lymphocytes (T and B cells) in separate tubes. Trying to do them all in one tube may be difficult, especially for all of those different cell types. Our lab stains 1 million cells per tube, and I think you should have enough for that.
Which T and B cell subsets are you interested in, besides Th1 and Th17 cells?
Hey Valerie,
I'm looking to perform a similar experiment using rat colonic tissues. Would it be possible for you to send me the protocol you've used in your lab?
Thank you,
Susheel
Hi Susheel,
I have attached the protocol below! Let me know if you have any questions.
Valerie
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