I have added anti- CD28/anti-CD3 beads to in vitro culture of murine T cells. Some of the cells have increased in size which indicates their activation. However, is there a method to quantify the activation of T cells? (I know IFN- gamma levels are measured but is it reliable?)
There are several readouts you can use for activation of murine CD8 T cells. Shifting of surface surrogates from CD44-CD62L+ to CD44+CD62L- is a simple readout that occurs quickly. You can also measure proliferation with Ki67 frequency or dilution of a proliferation dye (e.g., CSFE, stain before adding beads). More thorough indicators include IFNgamma, TNFalpha, IL-2, CD107a, and/or Granzyme B. INFgamma detection with ELISA or flow cytometry is a common and reliable metric for activated CD8 T cells.
Dear Abhay Kanodia
Check out my paper on activation and survival of CD8 T cells. There you find protocols for most of the assays that Brett P Gross mentioned, including protocols for surface antibody/ CFSE staining and flow cytometry etc.
Article A role for the histone H2A deubiquitinase MYSM1 in maintenan...
Please let me know, if you have any specific questions on how to set up the assays.
All the best,
Michael
@Brett P Gross and @Micheal Forster. Thanks a lot guys. This was a great help
Michael Förster I read the paper aand have the following questions:
a) Does the Cytofix/cytoperm kit (Becton Dickinson) provide any advantage over the conventional para-formaldehyde fixation?
b) Did you use brefeldin A or monensin before fixing the cells for granzyme B or IFN- gamma?
c) What was the antibody dilution you used for granzyme B and IFN-gamma?
Thank you!
Dear Abhay Kanodia
To a) I usually used cytofix/ cytoperm for intracellular stainings, especially intracellular cytokine stainings. PFA works very often equally well, however maybe not as good as the (expensive) BD reagents.
To b) For IFNg intracellular staining it does not matter, you can use either, for TNFa it does; there you should use Brefeldin A; for granzyme B you can use Brefeldin A as well.
To c) I strongly advise you to optimize the antibody concentrations for your specific flow cytometer/ settings cause there's wide variation depending on how well your instrument is maintained!
On a sidenote: The protocol and reagents for a Ki67 FACS stain you'll find here.
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I hope that helps!
All the best & good luck experimenting,
Michael
Hi, I want to activate CD8+ T cells purified from mouse spleen with CD3/CD28 dynabeads.
the protocol tells: bead-to-cell ratio of 1:1, and addition of IL2
did you try without IL2 addition?
Dear Sarah Amar
I briefly wanted to reach out concerning your question. I usually added IL-2 in T cell cultures, where I don't have a strong mitogenic stimulus (like plate bound anti-CD3), but rather coculture experiments with antigen specific T cells (from TCR transgenic mice). In that context you can 5 U/ml (being stingy) or up to 100 U/ml IL-2 in solution, which will increase cell viability and activation/ differentiation. Higher doses of IL-2 may favor T regulatory cell differentiation, but this is extremely context dependent, meaning you need to optimize it for your specific conditions and experimental questions.
I hope that helps.
All the best & good luck experimenting,
Michael
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