I have a protocol and experiment that calls for using 4.5 mg of collagenase. My scale only reads as low as 10mg. Will I be ok using 10 mgs when it calls for 4.5? Please don't say to measure 10 and half it. Just answer what would be the result of using 10mg instead of 4.5. Will my preadipocyte cell number be damaged? Thanks.
Which type of collagenase do you use and did you checked it with the type of collagen in your tissue?
My suggestion for this problem: use a lower concentration of collagenase and incubate a little bit longer. The cells are not that much stressed and you probably get more material.
I use collagenase from Clostridium histolyticum type IA (Sigma, C9891). Its activity is about 125 U/mg (collagenase) and about 350U/mg (protease). Solve it in TRIS 50mM, pH7.4 with 0.36mM CaCl2, centrifuge at 2500rpm for 20min and filter the supernatant it first through a 0.45ug filter and then a 0.2ug filter.
I use it to dissociate different tissues and, depending on the "hardness" of the tissue, this can take a few hours. In between I vortex the tissue very thoroughly. Further I admit, if the tumor tissue arrives from the hospital at Friday evening I add much more collagenase...
I never noticed that the cells are dying during this procedure. But this may depend on the sort of the tissue.
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