I'm interested in using Sircol Collagen Assay kit to determine if my stimulated renal cells are inducing collagen synthesis, does anyone have experience with this? i'm using adherent cells in culture, thank you!
Yes, the Sircol kit from Biocolor works very well on adherent cells. The first thing you need to know is that you should work in low serum because the kit will pick up protein/collagen in the medium.
Then, you can do two things if you like.
1. You reduce the amount of low-serum medium on your cells to just cover them for 24 hours. You take the conditioned medium from the cells and run that in the Sircol kit (instructions in kit). This will show you increases in soluble collagen that has not yet been cross linked.
2) You wash the cells 2X with PBS +Ca+Mg. It's really important that the calcium and magnesium are there. Then you either hypotonically lyse the cells, wash again, and scrape in acid to get the matrix off the plate - or you scrape the cells and the matrix in the acid. The protocol should tell you about the acid. If you lyse the cells, you get rid of collagen that isn't cross-linked into the matrix and/or is still in production in the cells. But you may not need to do that.
The important trick to the assay is actually when you are drying the pellets (after the sircol reagent is added) and removing excess fluid/dye from the tubes. That has to be done rigorously but carefully because it can give you a false positive if you leave some excess in the tube. You want it so that only the collagen/sircol pellet is measured once you solubilize.
Hope that helps. I think the kit works really well. If you haven't already, take the time to familiarize yourself with the basic story of collagen deposition and cross linking so that you know where you can expect to find your collagen. You could have more or less because the production of the protein changes, the secretion of the protein changes, the cross linking of the protein into the ECM changes, or the turnover of the extracellular protein (MMPs etc) changes.
Hi Mariya, I am running sircol assay and I have my samples (supernatant) frozen down in -80. This is possibly could produce crossed linked collagen, the company (biocolor) advised me to use a cold acid pepsin collagen protocol (0.1 mg/ml in acetic acid 0.5M) and keep it overnight at 4 C to release the collagen to the solution. how much do u think I should I add to my samples? I got 300-400 ul each sample.
Regards
Waseem
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