Hello. I would like to assess the cytokines produced by cryopreserved PMBCs after PHA stimulation. More precisely, I want to measure intracellular cytokines using flow cytometry (with PMA/ION) (IFN-gamma, TNF-alfa, IL-17) and those from the cell medium using ELISA (IFN-gamma, TNF-alfa, IL-10). These are my queries
1.- I think that after thawing the PBMCs an overnight incubation without stimulus is a good idea; is that correct?
2.- Which PHA is better: PHA-P or PHA-L? Should I add IL-2 to the medium?
3.- Based on the literature, I think that 48 h stimulation is enough. However, when should I change the medium? Just the first day (the first overnight without stimulus)? And, what happens between the 2nd and 3rd day? Should I add more complete growth medium?
4.- Any other comment about this kind of experiment?
Hello Maria Inmaculada Dominguez-Mozo ,
1. Many people incubate cells after thawing. The "resting" period can be anywhere between 2h and 24h. I haven't found a paper about this topic yet, but from what i've read and my personal experience, 2h is the minimum and you should never let them rest for more than 24h because they start dying without stimulation. I would say anything between 2-24h should work. I think it's more important to choose a number and stick with it for all the samples in order to avoid an additional source of variability.
2. I've never worked with PHA so I have no opinion on this.
3. I don't know about the duration of stimulation, but I can say something about the cell medium. If you go for a 24h resting period, I think it would be best to change the medium before starting the stimulation assay. After that, if your cell medium is well prepared, you don't have to change it until the end. PBMCs at 1-2 million/mL can survive in proper culture media for more than 48h. There's enough nutrients for them. We use RPMI supplemented with 10% FBS and 1% anti-mycotic anti-bacterial solution. We frequently do cell cultures with 2 million PBMC/mL and they are just fine after 48h.
4. I think there are multiple ways of going at this. I am not sure how exactly you are willing to stimulate the cells with PMA, but I guess it's after the 48h of incubation with PHA?!
A short example of a protocol
- thaw PBMC
- 24h resting
- change culture medium
- add PHA
- 48h incubation with PHA
- change medium (and save the discarded one for ELISA if you want to do ELISA for the cytokines released during the 48h of incubation with PHA???)
- add PMA/ION (+Brefeldin A?) and incubate for 5h
- change the medium
- surface staining (if needed)
- cell fixation
- permeabilization
- intracellular cytokine staining
- data acquisition on a flow cytometer
This is a summary only. If you want to take a peek at our intracellular cytokine staining protocol after PMA/ION/BFA stimulation:
Article Variability of ex-vivo stimulated T-cells secretory profile ...
Article Cytokine production in ex-vivo stimulated fresh and cryopres...
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