Tissue Digestion Kit

This kit is composed of recombinant collagenase mixed components and recombinant porcine trypsin. The recombinant collagenase mixed components are expressed in E. coli and are specific to the bond between the neutral amino acid (X) and glycine in the collagen-specific amino acid sequence Pro-X-Gly-Pro; the recombinant porcine trypsin is recombinantly expressed in Pichia pastoris and can specifically cleave the carboxyl-terminal peptide bonds of lysine and arginine in proteins. Both the recombinant collagenase mixed components and the recombinant porcine trypsin do not contain animal-derived ingredients, and can be used to efficiently digest animal organs such as the heart, liver, and kidney to obtain corresponding primary cells.

Instructions for use (digestion of mouse heart tissue, for reference only)

• Thaw the recombinant collagenase mixed components and recombinant porcine trypsin in the kit and mix them in a sterile state at a volume ratio of 1:4 to prepare a digestion solution. If further dilution is required, HBSS or PBS buffer can be used for dilution.
• Remove the mouse heart from the tissue preservation solution and place it in pre-cooled PBS (+2% blue chain double antibody) and wash 2-3 times.
• Remove the excess part of the tissue (such as connective tissue, adipose tissue, etc.).
• Cut the mouse heart into pieces (about 3mm3), transfer it into a centrifuge tube, add 1.5ml of digestion solution, digest at 37℃ for 20 minutes, and blow or invert the pipette every 10 minutes to mix well to fully digest the tissue. During this process, the digestion situation needs to be carefully checked to avoid over-digestion. If necessary, the digestion suspension can be examined under a microscope. It is best to observe more single cells or smaller cell clusters (<70µm) under the microscope.
• Let the digestion suspension stand for a while, collect the supernatant, add 1.5ml digestion solution to the precipitate, digest at 37℃ for 20 minutes, and mix by blowing or inverting with a pipette every 10 minutes or so to fully digest the tissue.
• Repeat the above steps once.
• Combine the collected supernatants, add 5ml DMEM complete medium to terminate the digestion, centrifuge at 4℃, 300g for 5 minutes, and discard the supernatant.
• Resuspend the cell pellet with 5ml PBS (+1% blue chain double antibody), pass through a 70µm cell sieve, and centrifuge at 4℃, 300g for 5 minutes.
• Resuspend the cell pellet with 5ml PBS (+1% blue chain double antibody), centrifuge at 4℃, 300g for 5 minutes, and discard the supernatant.
• Resuspend the cell pellet with an appropriate volume of DMEM complete medium and culture it in a cell culture dish.
  

If more cell clumps than single cells are required, shortening the digestion time or reducing the proportion of trypsin is recommended;
Try to avoid repeated freezing and thawing of this product. If there is any surplus, it is recommended to pack and store at -20℃