I am conducting invasion assays through collagen gel. The gels were successfully setting but they stopped setting. my recipe is:
500ul collagen (1.5mg/ml)
100ul 10x DMEM
400ul ddH2O
and approximately 15ul NaOH for adjust the pH.
I put 40ul in the 96 well plate, (either containing a chemokine or not) let it set for an hour at 37C then apply another 40ul and let it set for an hour at 37C. I chill all my ingredients, and I have tried wrapping the plate in parafilm to try and reduce fluctuations in CO2 levels
The only thing thats has changed is the bottle of collagen but my colleagues arent having an issue with their gels.
Any suggestions?
There are several modifications of Dulbecco's Modified Eagle Medium (DMEM), and the actual media selected may (possibly) deviate from the original formulation. It seems important to ensure that the pH buffering capacity is properly maintained once the original pH was increased by adding NaOH. Also determinant would be the concentration of CaCl2 at the media. You may want to check my answer at the following discussion: https://www.researchgate.net/post/Can_anyone_suggest_me_the_buffer_conditions_or_methods_for_solubilising_collagen_powder_other_than_acidic_condition
I extracted my own rat tail collagen and now the gel is setting fine. Still no idea why the collagen I had bought just stopped working
Probably denatured, or wrong concentration i.e. could have been a supplier issue (or storage at your end). Interested in how you prepared your rat tail collagen (acid or pepsin), you can easily isolate rat tail collagen with acid only i.e. crosslinks are acid labile, pretty unusual for tendon and you will retain telopeptide so this will affect fibrillogenisis; which method did you use
That is odd that it works with your own extracted collagen. What method of extraction did you use?
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