i would like to do surface immunostaining and live sorting of cryopreserved CD14+ human monocytes for further experiments. Does anybody have experience with this kind of experiments? Do I need to let cells rest after thawing or can immediately proceed to staining/sorting/recovering after sorting? Thank you!
Dear Sofya Perelman,
I think that whether you need to use directly or let them rest solely is based on the concentration of the cells. Do a cell count after you have thawed them. FACS studies usually use a very high cell density. If you think that the concentration is not sufficient, then plate them and let them become confluent.
Hello
i agree with Onur Kerem Polat , see these link
Hope help you
Good Luck
http://www.ufjf.br/ppgpmi/files/2012/08/3-criopreserva%C3%A7%C3%A3o-e-prod-citocinas-Ver%C3%B4nica-e-M%C3%A1rcio.pdf
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4249311/
http://iai.asm.org/content/early/2014/08/05/IAI.01973-14.full.pdf
I guess my main question was - are they likely to survive sorting immediately after thawing. I do not expect them to divide (become confluent) and my starting amount is 5mln cells.
Hi Sofya,
I would thaw them, wash them and count them. After that start the staining as you would normally do for flow experiments. If you want I could send you a protocol. I would definitely not culture them. Our lab is trying and we find that they (especially the non-classical subset) either die or differentiate after only 2 hours in culture. Regarding your question about sorting: you will have some cell death, but starting with 5mln cells you should have enough left. Which population exactly are you sorting, because you could consider a magnetic bead isolation. This is better for the monocytes.
Kind regards,
Anouk Hamers
Thank you! My experiment would require isolation of CD64+ cells and further differentiation into M1 macrophages. I have seen protocols which start from frozen PBMCs but never directly from frozen monocytes.
Anouk, would you mind sharing your protocol? Thank you!
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