FIbroblast culture - (Apr/27/2012 )

Dear all,
I am feeling trouble to make primary fibroblast from skin biopsy.
I had tried various protocol but not succeded.

plz Tell me how to prepare the fibroblast from skin biopsies.

It can be difficult to do so, but the general procedure is to take a biopsy, disrupt the tissue by some means (usuallymincing with a scalpel followed by collagenase or trypsin), placing resulting stuff into a cell strainer or straight into a culture dish. Leave for a few days to allow the fibroblasts to migrate out of the tissue pieces and then start to clean up the culture.

You may need to supplement with more FBS and use antibiotics to suppress the natural flora of the skin.

Once isolated and growing, what confluence is recommended? I have found I need to let them get to 100% confluence in a 100mm petri dish before I can get a decent amount of protein. Next I am going to try to use them in a flow cytometer and I'm worried that if they are at 100% confluence they may be too "clumpy" to run through. Does anyone have any suggestions/insight?

Most primary lines are contact inhibited and will stop growing (often irreversably) once confluent. If you need more protein, use more dishes. For the Flow, I don't think that the confluence will have an effect on the clumping, but it should have an effect on the proportion of cells in different parts of the cell cycle.