Hello Jin,

It depends on what exactly you want to achieve with this question. Typically, I would use CD3/CD28 to stimulate T cells over a few days or to keep cells in culture for a similar time period. In that time, I would expect that T cells will start to produce the aforementioned molecules. However, I would say this would not specific to T cells from tumor-bearing mice, but can also be observed in healthy/ naive mice. That is probably not what you want to see.

Of course, it is possible that cells from tumor-bearing mice are "pre-activated" and will start producing these cytokines sooner. However, I would recommend to first try a simple ex vivo staining (perhaps with restimulation).

Granzyme B can be detected in CD8 cells (and sometimes in CD4 cells as well) without restimulation; you can simply perform extracellular staining, fixate/ perm and perform an intraceullar staining. The clone GB11 works quite well.

For cytokines, I would recommend a restimulation. Here, after isolating the cells, you incubate the cells with a protein transport blocker which essentially "traps" the cytokines in the cell. Simultaniously, you can stimulate the cells with something like PMA (and, most of the time, Ionomycin) to induce furhter cytokine production and enrich the cells. This is quite toxic for the cells, so you might have to optimize the incubation time, but it is efficient to accumulate cytokines. An even better approach would be to restimulate your cells with a peptide mix specific for your model, but that is more complicated and PMA/Iono should work for a first experiment.

After this restimulation, you can stain your cells with TNFa/ IFNg/IL-2/GranzymeB.

Keep in mind that restimulation may decrease the quality of other stainings.

You should always have a reference control (here: cells from a healthy mouse) to get meaningful results.

Look for restimulation protocols, but in short something that works for me:

1. Isolate cells.

2. Seed 2E6 cells/ well in a round bottom 96 well plate.

3. Add 200ul RPMI+5% FCS + 1x Brefeldin, 1x Monensin, 1x PMA, 1x Monensin*

4. Incubate at 37 °C 5% CO2 for (up to) 5 h

5. Stain Live/Dead; extracellular; Fix/Perm/ intracellular**.

6. Analzye via Flow

*The are all often sold as concentrates by BioLegend, BD etc and are relatively cheap.

** Look for cytokine specifc Fix/ Perm / stain buffers from a company to mitigate this potential source of error instead of making your own buffers. 1 Set will be enough for 100s of stainings.

Hope this helps! :)

Best

Johannes

Johannes Brandi Hi Johannes, thanks a lot! Do you suggest adding Monensin or Bredfeldin? Or both? The main purpose for me is to check if a new drug can induce more cytokine-expressing CD8 T cells in the tumor microenvironment, so I would try your protocol.

Hi Jin,

we always use both, which worked well for us in the past. It might be a bit "over the top", but so far we got satisfactory results. I´d try both first and if you observe too much cell death, adjust the protocol. BioLegend has an overview on what they recommend for each cytokine:

https://www.biolegend.com/en-us/intracellular-staining-stimulation-guide

A few tips:

Perhaps you micht also want to stain CD107a (LAMP-1), a degranulation marker often used in combination with cytokines. If so, I´d recommend adding it to the medium in which you will incubate the cells; this will dramatically improve the staining over a surface staining after the re-stimulation. I suggest a dilution of ~1:100-1:200 in the medium which you will use. Because you might need a bit more antibody using this method, I´d try to use a cheaper fluorochrome for this marker, if avaialble (For example APC over BVs). Of course, prioritise your most important markers first if you don´t care so much about CD107a.

The restimulation will impact the expression of some markers; among them is certainly CD3. Some improvement can be gained by staining CD3 intracellular (also works better if staining ex vivo ;)), but it won´t be as good as a normal ex vivo staining. If antibodies are properly titrated, you should still be able to see T cells and if using CD8, you should be able to identify CD8 T cells fairly well. Just be aware.

Besides the cytokines, I would try to keep the panel as simple as possible to avoid potential problems. Wu usually have lineage marker, as well as CD44 included as an activation marker and maybe 1-2 markers of interest, but noch much more on the surface.

Best

Johannes

Johannes Brandi Thank you for sharing the helpful experience and tips! Will definitely give it a try.

Jin