i want use CD3/CD28 to measurement intracellular cytokines
someone has Protcol.please
Anti-CD3/28 priming of T cells is a standard tool for intracellular cytokine detection by flow cytometry. We typically use CD3/28 priming followed by short term restimulation in presence of PMA/Ionomycin/Brefeldin.
We use the anti-CD3/CD28 Dynabeads with Golgiblock and it works great. In our hands, we use a 1:1 bead to T cell ratio but you might need to optimize for your studies.
Yes- this is a pretty standard method for assessing intracellular cytokines in T cells. Key is to add the Golgiblock (or Brefeldin) to keep them from being secreted.
I'm also about to try this assay (with isolated T cells from mice splenocytes) and I have a few questions:
anti-CD3 should be plate-coated and anti-CD28 added soluble, just like an standard proliferation assay (I dont have beads)?
It is necessary the short term restimulation in presence of PMA/Ionomycin?
How long is it recommended to incubate before adding brefeldin?
Thank you in advance, I will really appreciate your help!!
i used PMA+ionomycin to stimulate T cells for 5 hours, and i used brefeldin for that last 2 hours. i used intracellular cytokines staining by cytometry to see the exprission of positive T cells.it is a powerful stimulation so you can see the augmentation just after 5 hours. For anti CD3/28 i think you can use it, but you will need to stimulate the cells more than 5 hours ( 24 or 48 hours). you can find many articles in this subject.
Jeremy Goettel, can I get in contact with you for more in detail protocol? I am planning to use CD3 CD28 dynabeads to activate immune cells isolated from adipose tissue. Did you try the beads for short-term stim? I want to look at IL-17A, INFg and IL-4
Hi All, I am trying to do the same here with PBMCs from cord blood and no result. Actually, I am using the Anti-CD3.CD28 dynabeads to stimulate the cells for 8 hours, and addition of Brefeldin A for the last 4 hours to prevent secretion of cytokines. Then we stain for surface markers before fix/permeabilization using FOXP3 kit buffer before staining for intracellular cytokines like IL-2, IL-4 and IFN-g plus TF like FOXP3. But no signal for these markers by flow cytometry. Any suggestion why? or to improve result. We usually keep the cells fixed overnight in 4C before intracellular staining the next day. Could this be a problem?
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