Hello everyone.
We have a vaccine that elicits a protective response against a pathogen in mice but we want to know which is the T helper type that has been developed (Th1, Th2 or Th3).
We want to perform an intracelular stain to study by flow cytometry the cytockines produced by the splenocites of the immunized mice but first we have to reactivate the CD4 from spleen.
We tried to cultivate the pathogen during 16 hours with splenocites to see if the APC of the spleen could produce the immunological presentation, but it wasn't enough and we didn't see the response.
I was wondering what could have been wrong, wasn't the time enough? Are we using a low quantity of cells? (we are staining 50 thousand cells).
I dont know if anyone could give me any idea.
Thank you very much.
1. How are you detecting presentation? If it's by T cell responses? 16 hours is not enough. What you are trying to do is antigen recall. Instead of using the live pathogen, I suggest you use a heat-killed version. To eliminate any issues that live pathogen can have on antigen processing/presentation.
2. To do intracellular staining by flow, at the last 4 to 6 hours of cell culture you need to add an inhibitor (such as Brefeldin A) to have enough cytokines trapped inside the cells to be detectable by flow.
Hello Mr. Shen-An Hwang, thank you so much for your response.
The bacterias that we are using are heat inactivated ones, as you recommend.
Also, we are using brefeldin A as you said. The positive control with PMA ionomicine and brefeldine A works propertly and we were able to see the cytokines.
Our problem comes when we try to activate cells with the bacteria.
As you said, we are trying to see T cell adaptative response.
We readead a paper where they stimulated cells 16 hours and it was enough for them. That is why we decided this time, but as you said is probably not eough.
So, could you please tell me how long do you recomend us stimulating cells in order to see cytokine production?.
Thank you so much for your time.
So... I used to do this with MTB... and we were looking at activation of memory, so I used 48 hours or 72hours.