I'm looking for a protocol to isolate T cells from a melanoma tumour. I've found several but in some they use collagenase type I and in others type IV. What is the difference? Should type I be used for some cases and type IV for others?
Collagenase type I (from Clostridium histolyticum) is a crude collagenase preparation that can be used for the isolation of primary cells or for tissue dissociation by enzymatic means. The preparation also may contain caseinase, clostripain, and tryptic activities. Collagenase type 4 is designed to be especially low in tryptic activity to limit damage to membrane proteins and receptors but with normal to above normal collagenase activity.
Type 4 is devoid of any tryptic activity and the proteolytic activity of the collagenase enzyme itself is more specific and the enzyme is definitely more pure it has specific and specialized uses where tryptic activity is not expected to interfere with the experimental conditions.
At least sometimes, this term refers to a collagenase such as MMP9 that can break down type 4 collagen, the one in basement membranes. This will cut along the basement membrane and separate epithelium from connective tissue, which is useful for making epithelial cultures free of fibroblasts. I don't know if melanomas make type 4 collagen but it seems unlikely - it's normally epithelial cells that make that.
If you want to preserve the surface markers of your lymphocytes I would recommend Collagenase IV because it has low tryptic activity (as Mr. Mandal wrote). It can be useful for FACS analysis, subpopulation isolations based on surface markers, etc. to keep them.
a lot of information about diff. collagenases you will find here http://www.worthington-biochem.com/cls/default.html
@ Raul
The thread below recommends collagenase I for lymphocytes and IV for myeloid cells.
https://www.researchgate.net/post/What_is_the_best_enzyme_mixture_for_tumor_dissociation
see Hanh's answer.
The first commercially available collagenase was offered by Worthington in 1959. At that time we offered one type of crude enzyme which we tested only for collagenase activity. Eventually, with the cooperation of many in the research community, four basic profiles were identified:
Type 1 containing average amounts of assayed activities (collagenase, caseinase, clostripain, and tryptic activities). It is generally recommended for epithelial, liver, lung, fat, and adrenal tissue cell preparations.
Type 2 containing greater clostripain activity. It is generally used for heart, bone, muscle, thyroid and cartilage.
Type 3 selected because of low proteolytic activity. It is usually used for mammary cells.
Type 4 selected because of low tryptic activity.. It is commonly used for islets and other applications where receptor integrity is crucial.
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