I have my own fibroblast cell culture, and I would like to know if those cells produce a certain ECM protein. It was suggested to me to try poly-L-lysine coating before cell culture, and then do the stain as I normally would. From what I have been reading about poly-L-lysin coating, this procedure is used to facilitate adherence of cells that usually come off with staining (please correct me if I'm wrong). Since I am trying to stain a protein that is out of the cells, my question is if the protein will still be there after the first washes? Has anyone tried this before? Is this the right way to do it?
Many thanks!
Ana
Hi Ana, fibroblasts will adhere to TC plastic in the presence of serum. Serum has vitronectin, which is the major adhesion factor for cells in TC.
Forget about poly-lysine. Cells adhering to vitronectin will not come off the plates unless they are treated with trypsin -EDTA.
Fibroblasts can produce copious amounts of ECM proteins (collagens, laminins, fibronectin, etc) that are mostly deposited on the basal surface of the cells.
By definition, incorporated ECM proteins can't be washed away with saline solutions. Otherwise they wouldn't be a part of the ECM.
Steingrimur's answer is correct. I just want to add that if you are plating these cells on coverslips in a well of a tissue culture dish, then two options for helping the cells adhere to glass is to coat with poly-l-lysine (high MW) and/or acid wash them. Otherwise, there is no need to do any coatings on the plastic itself.
Fibroblasts grow happily on tissue culture plates and do not was away. Also if you are planning to seed on coverslips use tissue culture grade/autoclave. If your planning to do staining on coverslips, seed 200 ul of cells + complete media directly on coverslips, allow it to adhere for 4-5 hr followed by adding completed media 500ul/24 well each or as necessary. Your will observe nice growth, as serum has lots of adhesive proteins.
For fibronectin, tenascin and other ECM i directly seedcells in 48 or 24 well plate and stain it. The results are amazing. Also for fibronectin and tenascin-c do check that antibodies binds to ECM component of the proteins.
Good luck
- Badges
- Science topic