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Cat. No.: CCH2-100 (for 100mg)

Cat. No.: CCH2-1k (for 1g)

Description

Collagenase, derived from Clostridium histolyticum, is a protease that specifically recognizes the Pro-X-Gly-Pro sequence and cleaves the peptide bond between the neutral amino acid (X) and glycine (Gly). This sequence appears frequently in collagen. Collagenase is the only protease capable of degrading the triple-helical natural collagen fibers extensively found in connective tissues.

This product is Type II Collagenase, with an activity of ≥125 U/mg solid, suitable for the dissociation of heart, thyroid, salivary gland, liver, bone, cartilage, and other tissues and cells.

Activity Definition

One unit of enzyme activity is defined as the amount of enzyme required to hydrolyze collagen at 37°C, pH 7.5 over 5 hours, producing 1 μmol of L-leucine.

Protocol

Add 1 ml of HBSS (Hank’s Balanced Salt Solution) containing Ca²⁺ and Mg²⁺ to 100 mg of collagenase powder in each vial. Gently vortex until fully dissolved, preparing a 100 mg/ml (100×) storage solution. Sterilize by filtering through a low-protein-binding 0.22 μm membrane, aliquot into small portions, and store at -20°C in a light-protected environment.

Before use, thaw on ice and avoid repeated freeze-thaw cycles. The commonly used concentration for tissue and cell dissociation is 0.5–2.5 mg/ml, while 1–2 mg/ml is typically used for cartilage digestion. The optimal working concentration should be determined based on specific experimental conditions or relevant literature.

1. Using a sterile scalpel or scissors, cut the tissue into 3–4 mm pieces.

2. Wash the tissue fragments several times with HBSS containing Ca²⁺ and Mg²⁺.

3. Add a sufficient amount of HBSS containing Ca²⁺ and Mg²⁺ to fully immerse the tissue fragments. Then, add collagenase to the desired working concentration.

4. Incubate at 37°C for 4–18 hours. Using a horizontal shaker and supplementing digestion with 3 mM CaCl₂ can improve efficiency.

5. Filter the dispersed cells using stainless steel or nylon mesh screens. Collect the dissociated cells for further use. For tissues that are not fully dissociated, add fresh collagenase solution and continue incubation at 37°C.

6. Wash the collected cells several times with collagenase-free HBSS.

7. Resuspend the cells in the culture medium, then count live cell density using an automated cell counter or other suitable methods.

8. Seed the cells in appropriate culture plates with a suitable cell culture medium.

1. Preheat HBSS containing Ca²⁺ and Mg²⁺ to 37°C, then add collagenase. Supplementing with 3 mM CaCl₂ enhances dissociation efficiency.

2. Perfuse the organ with the collagenase working solution at an optimized rate.

3. Filter the recovered perfusion fluid through stainless steel or nylon mesh to separate dissociated cells or small tissue fragments from larger tissue masses. If dissociation is incomplete, incubate the tissue with fresh collagenase solution at 37°C.

4. Wash the collected cells several times with collagenase-free HBSS.

5. Resuspend the cells in the culture medium, then count live cell density using an automated cell counter or other suitable methods.

6. Seed the cells in appropriate culture plates with a suitable cell culture medium.

Only for research and not intended for treatment of humans or animals

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