If I want to quantify inflammatory cytokines production by a a single cerebral cell type, like microglia, after brain ischemia, one way is to isolate and select thoses cells by flow cytometry, and then quantify cytokinic mRNA after protein extraction step. However, I suppose that removing the cells from their environment (brain) causes their activation, and therefore the transcrition of genes encoding pro-inflammatory cytokines. In this case, can this method be considered reliable? If not, what method can replace it? A microglia-cytokine co-labelling in histological slices is one solution, but I would like to know if other methods are also available.
Thank you for your advices,
Lucas Le Roy
Ex-vivo activation of microglia is indeed of concern. Try doing the whole procedure at 4 degrees C to prevent this (as described in https://www.ncbi.nlm.nih.gov/pubmed/30471926 ). Best of luck!
Elliot
Hello Elliot,
Thank you for your answer and for this interesting paper that I did not know. Working at 4°C seems to be mandatory to limit ex-vivo activation. I didn't think it would be possible to isolate cells without a collagenase / DNase step and with ice cold 40% Percoll but they seem to have succeeded !
Lucas
Lucas Le Roy I have also successfully performed this protocol without use of enzymes. You will want to use Ultra Pure BSA (free of nucleases)for your FACS buffer and probably also include RNAsin or other RNAse inhibitor throughout the protocol to prevent your RNA from degrading. Good luck!
E
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