Hi,
I want to ask will collagenase digestion of mice tissue affect the certain epitopes? I use collagenase D (1 mg/ml) digest the tissue for 1 h, will this affect some epitopes? if so, how to avoid it? Thanks!
Xin
The most common collagenase products are sold by Sigma, Worthington, or many other companies. These products (including Collagenase D) are minimally purified Clostridium histolyticum culture supernatants that contain collagenase and neutral protease activity. They are referred to as crude or enriched collagenase since biochemical characterization is limited to several enzyme activity assays. It is likely that the protease (neutral protease and clostripain) in these products may affect epitopes on CD45 (not the collagenase since it has a very narrow specificity). You need both these activities to release cells from tissue. Collagenase alone is insufficient for cell release, so you must have the enzyme mixture also contain a neutral protease to accelerate degradation of the extracellular matrix.
An approach to overcome this problems is to use a similar concentration of your current lot of collagenase on a leuckocyte containg population and determine if the CD45 epitopes are lost. If not, go ahead and use it. If you do get a loss of epitopes then either titrate down the enzyme and see if you can retain the epitope or use purified collagenase and purified neutral protease. First, determine if thermolysin, or a dispase equivalent enzyme (BP Protease) digests the CD45 epitope (best to do a titration). Assuming the CD45 epitope is intact, then use the purified enzymes to digest the tissue..
Hi Robert,
Thanks for your detailed information!
Digestion solution for colon tissue, I use
1. Collagenase D, Roche Diagnostics (1 mg/ml)
2. DISPASE II, SIGMA (1 mg/ml)
3. DNase I: 20 μg/ml
I lost many CD45 positive cells by using this digestion,while for splenocytes without digestion, I get the large amount of CD45 positive cells. I think the digestion time is too long, and casue the CD45 epitope lose.
Another auestion is that why people use Ca+ Mg + free PBS as wash and digestion buffer? Will Ca+ Mg + also influence the epitope ? Thanks!
Hi Ibrahim,
Thanks for your message!
If Collagenase D do not affect the strcutures and antigenicity of CD45, will DISPASE II and DNase I affect ? they are all in my digestion solution, I think the digestion time is the main factor for my sample, since lymphocytes expose too long in digestion solution, and may affect the CD45 epitope, while CD11b, CD 11c expression is OK.