I am trying to design an experiment in which I desire to test if my compound would alter T cell differentiation (Th1, Th2, Treg etc). In this experiment I intend to utilize macrophages (and not DC) since it suits more to what I see in vivo. Even tough I found some articles in which they utilize protocols similar to what I need, I only found analyzes of T cell proliferation. I would like to know if in an in vitro setting, with certain stimulation, if I can coculture T cells and be able to differentiate them by FACS.
Dear Juliana,
in your experiment settings, do you sort T cells or Tregs separately to stimulate the cells with coculture with macrophages? if you don't intend to do so , I suggest that:
- You isolate T cells and macrophages from your in vivo mice experiment ( stimulated vs non stimulated).
-You coculture T cells with stimulated macrophages and non stimulated macrophages.
-You must add certain cytokines as IL-2, GM-CSF for example to maintain T cells viability in culture ( you need to optimize the concentration and ratio T cells/Macrophages).
-You can freeze the medium of cell culture for cytokines measurement later on as marker of differentiation. And look at the cells by facs using a panel of T cells markers and transcription factors.
*Ideally, I will stimulate each population of T cells individually specially Treg cells but all depends in you project purpose and scientific questions behind.
Good luck.
Dear Juliana!
https://www.researchgate.net/topic/T-Cell-Differentiation
Dear Juliana Del Sarto
Depending on your access to certain equipment/ facilities, I would suggest that you assess cytokine production (by ELISA, Luminex or intracellular cytokine staining/ flow cytometry), expression/ upregulation of costimulatory molecules on both macrophages/ T cells and induction of certain signature transcription factors (e.g. FOXP3 for Tregs etc.) to assess you cocultures, in addition to T cell proliferation (by thymidine incorporation or CFSE dilution).
For some protocols and reagents, please see attached for reference.
Article A role for the histone H2A deubiquitinase MYSM1 in maintenan...
All the best & good luck with your experiment,
Michael
Fadi Jebbawi ,
greetings and best of luck with your experiments.
I think Michael Förster gave an already seemingly complete answer.
If you are not sure about your protocols, don't hesitate to ask. Keep in mind, that especially the assesment of cytokine production can be tricky (type of stimulation or unstimulated, intracellular cytokine assesment vs supernatants etc...)
Good luck and all the best!
Robert
Robert Ose,
Thank you and good luck for you too in your experiments.
There is nothing called complete answer till you have the results for your experiments . I see your comment in this section is just to make a comment without any suggestions for the scientific question that Julianna is asking. (Irrelevant)
I am not sure that you will be helpful in the protocol part as already I have more then 10 years expertise working on T cells biology. Cytokines production is not tricky once you have the experiment settings tested and validated in the lab with standard protocols.
Meanwhile good luck in your ''Thesis'' …..
Best,
Fadi
Fadi Jebbawi I'm sorry mate! I just wanted to answer to Juliana Del Sarto and mixed up the mentions. Didn't want to critize you or your answer at all.
As I said, your name accidently slipped in my message instead of Juliana. I just wanted to support the answer of michael.
no offense intented
all the best!
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