I will do cell culture with specific patients PBMC. I isolated the PBMC and now will stimulate them with PHA as I want to see Th17 association with the disease. After stimulation I will take the supernatan and assess with cytokine ELISA. What must be the cell counts per well for 96 well plate, 24 well plate. What must be the concentration of PHA? And how long must be the stimulation? Is PHA solely enough for the stimulation for cytokine expression? Anybody have any suggestions.
I've used PHA as a positive control for IFN-y ELISpots. Usually I get a very high response using 100,000 - 200,000 cells with a concentration of 10ug/ml in an overnight assay. You could start at 10ug/ml and perform a serial dilution (or increase). I'd keep to 1 to 2 million cells per ml of media. If this is going to be a positive control for your assay, look at SEB as well, or spike a non-stimulated control with the a recombinant version of the cytokine you are targeting with a known amount. Another great idea is the CBA kits from BD, if you have access to flow, you can target a number of cytokines in one assay, I've used the TH1/TH2/TH17 kit and it worked great.
Thank's Craig. I will stimulate all the PMBC of patients to investigate cytokine profile after nonspesific stimulation but in parallel there will be PBMC of patients which will not be stimulated. I am thinking to do a 100.000 cells per well of a 96 well plate or 1 million cells/well for a 24 well plate. In articles PHA is used 1-5 microgram/ml. I will stimulate 72 hours. After stimulation I will take the supernatan and use ELISA's for IL-23, IL-12/23P40, IL-17AF and IL-35 to investigate the relationship of Th17, Treg with the disease. Does the method sounds good?
Sounds good, I'd use 2 million cells per mL in 24 well plate. IL-6 may also be a target, some people suggest IL-6 is key in either switching nTreg to TH17 cells or both iTreg and nTreg to TH17 cells, depending on the papers you read. Remember phenotyping the cells via flow pre and post stimulation may be worth doing as well.
Thank's a lot Craig for your suggestions. Good idea. You mean final concentration 2 million cells and final concentration 10ug/ml for PHA. So when I use 1ml cells (2 million) and add 1 ml PHA. Total volume of the well will be 2 ml and the final concentration for cells 1 million, and 5 ug/ml for PHA. How can I design it?and what do you think about cell numbers for 96 well plate and 48 well plate. Is there any standart for the number of cells when used these plates.
I'd use 2 million cells total, so yes the final concentration is A million per mL, and the PHA concentration of 5ug/ml should be fine if its been suggested in the papers you've found. I'd stick to 24 well simply because you have more supernatant to play with, you can freeze what you don't use at -80'C. Typically I work on a concentration of 1-2 million per mL, so in a 96 well plate if you add 200ul per well at a concentration of 1-2million cells per mL that would be 100,000-200,000 cells per 200ul.
Thank you very much Craig. Your answers were very usefull for me. I will be in contact with you later on. Best.
Hi, FYI, we cultured PBMCs at a cell concentration of 1 million cells per ml and also stimulated with PHA at a final concentration of 10 ug/ml. It worked for us to do cytokine ELISA for TGFB and IL10. Though not sure of cytokines associated with TH17, i have read papers which suggests that at this concentration PHA stimulation would be sufficient to analyze certain cytokines you are looking for. Good luck for your experiment.
hi, I used PHA as positive control for IFNg cytokine in whole blood assay. It should work in PBMCs as well. the concentration I used was 5ug/ml as final concentration. since it is a T cell mitogen, you should get response for Th17 as well. if you have access to folwcytometer then you should use BD CBA kits for Th1/Th2/Th17.
Craig Michael Rive I am doing IFN-Y intracellular staining in PBMCs and I'm trying to use PHA as a positive control. What brand and type of PHA do you use?
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