Hi, would you mind if I ask what is your activation protocol? Basically, you can use two blockers for cytokine secretion: brefeldin A and monensin. Brefeldin A seems to better work, but do not culture your cells with Brefelding A longer than 12 hours. Going back to your question, do you test re-call response? Do you use transgenic TCR T cells?. Anyway depends on what you want to figure out, you will need a positive control for your stimulation, i.e. T cells stimulated for PMA and ionomycin for 5-6 hrs.

Assuming your initial activation stimulus is a good IFNg inducer then no, you don't need to add an extra stimulus. At two days, your cells should still be producing IFNg from the initial stimulus. You can add Brefeldin A for 18 hours at a low concentration (e.g. 3ug/ml - I don't know what this equates to in terms of GolgiStop) to allow enough cytokine build up in the cell to get good staining.

Hi Jiawu. First, I note that Grzegorz`s experience in use of internal staining is rather more extensive than mine, so I`m not sure I can give you any information you don`t already know. Short answer to your question is no. Dr. Fadia Mahmoud (1st author on the paper you have) and I never needed to enhance T cell activation beyond initial stimulation with PMA/I or a lectin to "goose" expression of our cytokine(s) of interest at levels needed to test whatever hipothesis we were testing. I agree with Gzegorz`s assessment that BfA is superior to Mnsn - this based on my observation of clarity & reproducibility of our FACS results. Is this helpful?

HI. This is a very interesting issue. First, I would make a clear-cut distinction between ex-vivo and in vitro assays. Ex-vivo lymphocytes (at least in pigs) are often amenable to direct assays of some intracellular cytokines like IFN-alpha and IFN-gamma. Instead, during in vitro culture of lymphocytes stimulation followed by Golgi stop is often mandatory. In our experience, no further stimulation is needed at the time of Golgi stop. As for IFN-gamma, a convenient positive control is represented by PHA-stimulated lymphocytes after 40-48 hours in culture.

Thank you all.

My previous stimulation process was 1ug/ml soluble anti-CD3 for >2 days, then GolgiStop (monensin) for 4hrs. I used BD kit to do the staining. The result is not good.

Now I plan to use 3ug/ml ConA for initial 2 days and 3ug/ml soluble anti-CD3 for 5hrs with GolgiStop. Hope this time works better.

It's a pitty at the moment we don't have PHA, PMA, ionomycin in hand.

Hi everyone. Just wanna follow up my first question. What is the best activation time for re-call response? I have antigen specific spleen. If I do ex vivo stimulation with the specific peptide, how long should I use? Thank you all.

you will need a longer time for antigen--also should add CD28 and CD49d--I beiieve for costim.

If you are using peptide you should have APCs in your system (monocytes or pre primed DCs), plus CD28 and CD49d. Usually you need to wait 10 - 12 hours, the BFA should be added at the last 8 hours. Your negative control should be the stimuli whithout the peptide.

Going back to your question concerning IFN-gamma measurement I strongly recommend ELISPOT instead of intracellular staining. This assay is more sensitive and pretty easy.

Mouse splenocytes respond quite well to ConA stimulation with regard to production of IFN-g. I used ELISPOT assays to determine the number and frequency of cytokine-producing cells. In some systems, IFN-g production doesn't necessarily need exogenous additional stimulation (no ConA needed)--I used it only as a positive control for my infection model. ELISPOT assays are no more difficult than ELISAs and work on the same principles.

ELISPOT might be a good alternative. However, if you would like to look at T cell subsets (and want to identify them with surface markers for flow cytometry), I would still give some effort on the intracellular staining. Because it should work.

It might be better to use Golgi Plug (Brefeldin A) rather than using Golgi Stop (Monensin). I just checked and found this paper showing that Brefeldin A gives better results (although IFN-g is not in this paper):

Cytometry. 2001 Jun 15;46(3):172-6. Evaluation of monensin and brefeldin A for flow cytometric determination of interleukin-1 beta, interleukin-6, and tumor necrosis factor-alpha in monocytes.

Schuerwegh AJ, Stevens WJ, Bridts CH, De Clerck LS.

Good luck,

For Ex vivo stimulation you can stimulate splenocytes by adding peptide (~10uM) to the culture for 6 hrs or so with Brefeldin A for last 3 hours. You don't need to add APCs the APCs in the splenocytes are sufficient to present antigen. I use it routinely and works really well.