Looking at the DataSheet of the collagenase for fat digestion in order to extract adipose-derived stem cells. It is said to dilute 1-2 mg of collagenase in TESCA buffer (ontaining 50 mM TES, 0.36 mM Calcium chloride, pH 7.4 at 37°C).
My purpose is the digestion of fat to extract the cells from the stromal vascular fraction. So I do not know if I can dilute the collagenase directly in DMEM medium because I am not going to do a metabolic studio or something like that.
I cannot find any informmation about that in protocols or publications because they are not so detailed (they only say to digest the fat in a solution of 0.1 mg/ml of collagenase in DMEM, 15-30 min at 37ºC).
If I am not wrong I do not have to use Serum in the collagenase solution right? and antibiotics? Protocols present highly variations.
Thank you very much,
Sergio.
No, you do not need (or want) to add serum in your digestion reaction mixture.
Collagenase requires Ca++ for its activity and pH is important. Check the composition of your medium to ensure that Mg ion concentration is considerably lower than Ca++.
If you are timing the reaction by observing the appearance under a microscope, you can add serum containing medium to slow the activity of Collagenase and increase the concentration of Mg++.
I use HBSS containing Ca and Mg at a final concentration of 0.075%. You can add 2% BSA. Sometimes I also use 1% pen/strep. Make fresh solution just before digestion!
Yuri Ciervo can you please elaborate on the need of 2% BSA in Collagenase digestion.
It limits cell damage from the enzymatic digestion. Especially if the digestion goes for more then 1 hour.
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