I have cultured endothelial and smooth muscle cells from the aorta of rabbits. There has been an issue with fibroblasts in my cultures out growing cells. Is there and easy or cheap ways to fix this issue?
Thanks
Phil
Hi Philip,
in our lab inorder to avoid fibroblast contamination we use to preplate culture solution for 1-2 our in normal plastic, (fibroblasts quickly adhere), and then harvets the supernatant for target cells plating.
Good Luck
Try to eliminate fibroblasts by panning onto a Thy-1 coated substrate, immediatly after having prepared your suspension.
Phillip, I hve tried 2 methods to control fibroblast with success. Plate the endothelial/muscle cells with fibroblast for 1 hr. Then aspirate the media into a fresh cell culture flasks. Fibroblast will adhere to the plastic in the first cell culture flask, you may need to this more than once. A second method after aspirating the media from the original flask after 1 hr, decrease the concentration of the FBS to 1-5%. Endothelial cells will proliferate at this concentration of FBS. I suspect muscle cells will also proliferate at 5% FBS.
A third method which will deliver cleaner cultures is remove the media, add PBS to remove FBS proteins, aspirate PBS, add 1% solution of EDTA:Versene for 3 minutes. Fibroblast will lift off plastic, endothelial cellsremain attach. Aspirate EDTA:Versene, rinse with PBS, then add media with low FBS concentration. Essential point is limiting the EDTA:Versene to 3 minutes.
I would mean in my first comments "onto an anti-Thy1 antibody- coated substrate". We use with success this méthode to eliminate fibroblasts from dissociated sciatic nerve, in order to purify Schwann cells.
- Badges
- Science topic
- Similar topics
- Biological Science
- Cell Biology