Collagenase question - (Mar/14/2007 )

Hello All,

I have to use collagenase for digesting heart and lung tissue to isolate endothelial cells. One paper sugegsted the use of Type A collagenase while the other mentioned the use of type IV A.

I am not sure of the difference between the different types. I looked up on the sigma aldrich website and found that certain collagenase are suitable for isolation of specific cell types, but nothing was mentioned for endothelial or vascular cells.


Could someone give me some insight on this.

thank you

Pooja

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Hi! Sorry, May be it is not answer for your question but I would like to note, when I work with isolation of endothelial cells from umbilican vein I used more soft ferment dispase because of less contamination of SMC and fibroblasts in my endothelial cells . Collagenase iV type is "hard" ferment and stripp all other cells of connective tissue ( of course you can select conc and time incubation) . But in my case I cannuled vein ( source - Human, not mouse). If you have no possibility to do so may be it does'nt matter ( I mean difference betw collagenase and dispase). may be with collagenase it will be better because you should high quality digest connective tissue

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if you there is no going protocol try to optimize or establish aprotocol for your need; collagenases even same types differ between companies and lots; try alternatives as suggested; pronase may also an alternative; also, try mixes of proteases; very important beside type and conc is time if incubation

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Hi,

Thanks for all the suggestions. I read some recent articles where the authors used collagenase A. I have ordered it from Roche and will try and see if that works. if not I will alter the incubation times.

Bests

Pooja:)

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hi There,

I know its been a while since I last replied. i contacted someone at harvard and their protocol called for the use of collageanse Type 1. Since then I have been using the sme from Worthington. The protocol seems to be working well, besides the fact that after I filter the crude cell suspension throught the 70 micon nylon mesh, my filtrate is slimy.

What could be the reason for that?

Thanks a lot for your help

Bests

Pooja

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It sounds like DNA released. Do you see cells entrapped by these glue like substance? if so DNAse may help. What organ/ tissue/animal species are you working with?

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