Type I and II collagenase Mix preparation
Materials and Reagents
- HBSS with Ca2+ and Mg2+ (Gibco 324020)
- type 1 collagenase (Worthington biochemical LS004194) 1 vial
- type 2 collagenase (Worthington biochemical LS004174) 3 vials
- 1mL Luer Lok seringes (BD #309628)
- 20G needles (BD #305175)
- 15 mL tubes
- 50 mL tubes
- 150mL filtration unit Stericup 22m (Millipore ExpressTM plus 0.22m #
SCGPU01RE).
Enzyme reconstitution:
- In order to reconstitute the lyophilized enzyme inject 1mL of HBSS through
the cap of each vials and vortex them.
Type I collagenase:
- Open CAREFULLY the Type I collagenase vial and transfer its content to a
clean labeled 50mL.
- Rinse the Type I collagenase vial with 1mL of HBSS and transfer it in the
50mL tube.
- Repeat this step 3 additional times in order to obtain 5mL of enzyme
suspension.
- Bring the volume up to 24 mL by adding 19mL of HBSS.
Type II collagenase (for each vial):
- Open CAREFULLY the Type II collagenase vial and transfer its content to a
clean labeled 50mL.
- Rinse the Type II collagenase vial with 1mL of HBSS and transfer it in the
50mL tube.
- Repeat this step 3 times in order to obtain 5mL of enzyme suspension.
- Proceed identically with the second and third vials in order to obtain 15ml of
the type II collagenase suspension.
- Bring the volume up to 24mL by adding 9mL of HBSS.
Enzyme cocktail stock solution:
- Mix together the 2 enzyme preparations (final volume 48mL).
- Filter the enzyme cocktail through a 22m Stericup.
- Distribute the enzyme cocktail stock solution in 3mL aliquots.
For final use, this enzyme cocktail stock solution has to be diluted by addition of
22mL of HBSS for a 25mL final volume.
Updated 10-15-2014 by CM