I collected the blood sample(20ml) and i used 10 ml of blood diluted in 10 ml pf PBS.I diluted the rest of the blood with RPMI (supplemented with 10%FBS and 5ml penicillin/streptomycin) and i stored it at 7oC. I wonder if i can use it in order to isolate PBMC.Do the antibiotics or FBS affect the isolation?
I think that should be a problem. But you may take care that the solutions are all at room temperatur while isolation.
It would work fine for isolation: diluted blood is recommended when performing gradient separation. I do not a have a reference but I know it empirically. However, depending on the time stored, storage conditions might alter PBMC features such as viability, cell counts, number of adherent cells; and also downstream analyses like gene expression and cytokine production.
FBS should not be an issue but it has been reported that b-lactamic antibiotics downregulate production of some inflammatory cytokines.
With all this in mind take your best choice. Good luck
www.researchgate.net/post/Why_should_one_dilute_the_buffy_coat_before_ficoll_separation
More empirical info on PBMC separation xD
Thank you very much! Eventually, everything was ok with the isolation. This was just a trial. For tha final experiment I'm going to dilute the blood 1:1 with RPMI (non supplemented) . Now, I would like to ask you something else. According to papers, i add 150ul PBMC, 20ul thymol and 30ul LPS. After an incubation of 16 hours, supernatants are harvested and strored at -80 until analysis for cytokines. My query is whether I have to centrifuge the plate or not. Also, when it says harvest supernatants how many microliters I have to collect from tha 96-well plate?
Lymphocytes are not adherent cells so they remain in suspension. So, for cell-free supernatant harvesting you will have to centrifuge the plate. Depending on the use you might have for the remaining cells would be the speed you should centrifuge the plate. If no further use try above 2000 rpm.
Regarding the volume of supernatant, it will depend on how much is required for the ELISAs (for kits the required volume may vary from 50 to 150 uL). In order to avoid cell resuspension I wouldn't take more than 3/4 of the total volume (150 of 200 ul, in the example you mention). If you need to perform more than one ELISA you can always dilute the supernantant in dilution buffer (it is supplied in the kits).
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