Collagenase is a protease derived from Clostridium histolyticum. It specifically recognizes the Pro-X-Gly-Pro sequence and cleaves the peptide bond between glycine (Gly) and glycine (Gly) in this sequence, and this sequence occurs frequently in collagen. Collagenase is the only protease that can degrade the triple-helical natural collagen fibers widely present in connective tissue.This product is Type IV collagenase, with an activity of ≥125 U/mg solid and low trypsin activity, making it particularly suitable for islet cell preparation.
Usage Instructions
Ⅰ. Preparation of Collagenase Stock Solution
Add 1 ml of the appropriate buffer (determined according to specific experimental conditions or relevant literature) to each tube containing 100 mg of collagenase (e.g., HBSS [Hank's Balanced Salt Solution, containing Ca²+, Mg²+, etc.]). Gently vortex to dissolve thoroughly and prepare a 100 mg/ml (i.e., 100×) stock solution. Filter-sterilize using a 0.22 μm filter with low protein binding properties, aliquot into small portions, and store frozen at -20°C in the dark.Thaw on ice before use; avoid repeated freeze-thaw cycles. The typical working concentration for tissue and cell dissociation is 0.5~2.5 mg/ml, and 1~2 mg/ml for cartilage digestion. Determine the optimal working concentration based on specific experimental conditions or relevant literature.
Ⅱ. Tissue Isolation
- Use a sterile scalpel or scissors to cut the tissue into 3~4 mm tissue pieces;
- Wash the tissue pieces several times with the appropriate buffer (e.g., HBSS);
- Add sufficient buffer to submerge the tissue, then add collagenase to the required working concentration;
- Incubate at 37°C for 4~18 hours. Using a horizontal shaker and supplementing digestion with 3 mM CaCl₂ can improve digestion efficiency;
- Fully digested tissue can be filtered through a stainless steel or nylon mesh sieve and collected for later use. For incompletely dissociated tissue, add an appropriate amount of fresh collagenase working solution and continue incubation at 37°C;
- Wash the collected cells several times with collagenase-free buffer;
- Resuspend the cells in cell culture medium, and count the viable cell density using an automated cell counter or other methods;
- Seed the cells on cell culture dishes using a suitable cell culture medium.
Ⅲ. Organ Perfusion
- Add collagenase to the appropriate buffer (e.g., HBSS) preheated to 37°C; supplementing with 3 mM CaCl₂ helps improve isolation efficiency;
- Perfuse the target organ with the collagenase working solution at an optimized rate;
- Pass the recovered perfusion solution through a stainless steel or nylon mesh sieve to separate dissociated cells/small tissue fragments from larger clumps. Incompletely dissociated tissue should be further incubated with fresh collagenase working solution at 37°C;
- Wash the collected cells several times with collagenase-free buffer;
- Resuspend the cells in cell culture medium, and count the viable cell density using an automated cell counter or other methods;
- Seed the cells on cell culture dishes using a suitable cell culture medium.
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