I'm trying to culture eosinophils (from mice) in a 12 well plate in order to extract all proteins after 24h. I use RPMI 1640 without glucose. do I need to add glucose? What does everyone else do in order to keep them alive cause I don't seem to have enough proteins after bradford and I tried to double the amount of eosinophils in a well but still got the same results? Are they dying? What do I need to do in order to keep them alive?
Hadas :)
Dear Hadas,
what kind of mouse eosinophils are they (bone-marrow derived, from IL5tg mice, ...)? Granulocytes (including eosinophils) die quite quickly ex vivo. This can be prevented by addition of cytokines such as IL-5, IL-3 or GM-CSF. However, this will also activate the cells and might interfere with your experiment. Since I don't know the details of your experiment, it is hard to give advice, but maybe a shorter culture time could be a solution? Do you have FCS in your medium? And beta-mercaptoethanol? Glutamine?
Hi Christina!
thank you for the quick answer!
since i need to take samples through out the day (24h) i have to make them stay alive somehow, i cannot culture them for less time than that....
anyway, we add 10% FCS and 0.24% antibiotics.
I can add IL5 but if it activates the cells and makes them degranulate, it might be a problem, i need to take that into consideration.
anyway, other than that, what else i should have added?
should I have added more antibiotics? glucose (since RPMI doesnt have it but it does have glutamine)? anything else?
why do you add b-mercaptoethanol?
Hadas :)
I don't think that the antibiotics influence your cells' viability. For your question for b-ME you can also have a look here: https://www.researchgate.net/post/Has_anyone_used_2-mercaptoethanol_in_their_cell_cultures
IL-5 activates the cells but doesn't induce degranulation. However, of course it will influence gene expression.
How many cells do you have per condition?
we dont count them,
we pull out whole blood from ~50 mice, we separate PMNC with histopaque and RBC lysis protocol and then we make a pool (9ml RPMI) of all separated cells and divide them into 8 wells each containing 1ml RPMI + cells (1ml is "0" time of the experiment). then we collect the wells every few hours for 24h.
after how many hours do they start to die? because when we needed RNA it was fine enough what we had but for protein this is not enough...
Hadas :)
Dear Hadas, I recommend counting the cells before plating at least 1 Mio cells/condition. This should give you enough for a Western blot. Also, please keep in mind that PMNC contain neutrophils as well as eosinophils. The easiest way to check your eosinophil vs neutrophil content is to do a cytospin followed by e.g. DiffQuick staining.
Good luck
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