Please help with Collagenase Type I - (Nov/17/2005 )

I am trying to count cells that are entrapped in a collagen type I matrix. For that I need to digest the collagen first. I bought collagenase type I, clostridium histolyticum, 500units/mg. I was wondering what concentration of collagenase shall I add to my samples? And is there an optimal incubation ti,e? If I leave my samples for extended periods would that affect the DNA count? I presume it should not but I am not sure
Thank you

Rita,

I used to do this a lot (counting cells grown in collagen gels). I have a paper published in the journal Prostate (Barrett et al, 2005).

You should incubate the cells/collagen at 37 degrees C with the culture plate on a minishaker at very slow speed (circular rotation is good). I can't recall the units of collagenase type I that I used, but I made a 0.2% solution of it in media with 10% serum (so cells would be happy) and used about 200 ul for a 500 ul collagen gel volume (that's the volume of liquid collagen gel mixture that was orginally added to the plate).

I would incubate the cells/collagen with collagenase type I for about 1 hour to ensure full dissociation of the collagen gel. After this incubation, cells will need to be collected, centrifuged to remove media, and trypsinized prior to cell counting. I have a protocol that I used on a regular basis, if you or anyone else is interested.

Hi,

I would be interested in seeing your protocol for releasing cells from collagen gel with colagenase. I need to harvest viable keratinocytes from organotypic models made with type 1 collagen gel (rat tail). Would you be able to post the protocol please.

Many thanks,
Hayley

I am trying to count cells that are entrapped in a collagen type I matrix. For that I need to digest the collagen first. I bought collagenase type I, clostridium histolyticum, 500units/mg. I was wondering what concentration of collagenase shall I add to my samples? And is there an optimal incubation ti,e? If I leave my samples for extended periods would that affect the DNA count? I presume it should not but I am not sure
Thank you

Hi Hayley,

The protocol is attached. Let me know if you have any questions. Concentration of collagenase 1 could change based on number of units/mg supplied, but using too much shouldn't be harmful, since the collagenase 1 is specific for collagen type 1. If you want to obtain single isolated cells, you may want to add in another enzyme with the trypsin, such as dispase.

Best of luck.
-Jeff

Hi Jeff,

Many thanks for posting the protocol, it is very helpful. I just have one quick question..... I have been told to add 5mM of calcium into the collagenase buffer to activate the collagenase. Your protocol does not mention this - do you rely on the calcium already present in the medium for this?

Thanks,

Hayley