Hi,
Im trying to isolate dermal fibroblast for RNA.
I used collagenase, and than stained the cell to PDGFRa in order to sort the fibroblasts, but I get low number of live cells.
I succeed to sort only 4000 cells.
the isolation was from moues ears (4 mice), is there is better way? or good protocol for someone?
Thanks a lot
Shani
Hi Shani,
If you want to isolate dermal fibroblast, I suggest you use mouse skin (abdominal skin). You can get around 12 cm2 and isolate around 600 000 fibroblasts / mesenchymal cells per mouse. Here is my protocol to isolate primary fibroblasts (not by adhesive in vitro culture). In this protocol, I used SCA-1 marker as positive marker for fibroblast/mesenchymal cell and a panel of negative markers to exlcude hematopoietic, endothelial and epithelial cells (CD45, TER119, CD11b, GR-1, CD2, CD4, CD41, B220, CD31 and Epcam). Using this protocol, we have been able to isolate RNA and perform RNA-seq on 3 biological replicates. If you have any question, don't hesitate.
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