SKU: 6609012859

Tissue Digestion Kit II

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Description

Tissue Digestion Kit IIProduct Specification Purity 95% by SDS PAGE Endotoxin <10 EU mg Stability & Storage This product should be stored at 20 and can be stored for at least 12 months. Avoid repeated freezing and thawing Background This kit is composed of a recombinant collagenase mixed component and a heat resistant metalloprotease. The recombinant collagenase mixed component is expressed in E. coli and has specificity for the bond between the neutral amino acid (X) and

Product Specification


Purity ≥95% by SDS-PAGE
Endotoxin <10 EU/mg
Stability & Storage

This product should be stored at -20℃ and can be stored for at least 12 months.Avoid repeated freezing and thawing

Background

This kit is composed of a recombinant collagenase mixed component and a heat-resistant metalloprotease. The recombinant collagenase mixed component is expressed in E. coli and has specificity for the bond between the neutral amino acid (X) and glycine in the collagen-specific amino acid sequence Pro-X-Gly-Pro; the heat-resistant metalloprotease is recombinantly expressed in E. coli and can hydrolyze the protein bond at the N-terminus of the hydrophobic amino acid residue. Both the recombinant collagenase mixed component and the heat-resistant metalloprotease do not contain animal-derived ingredients. At the same time, they can efficiently digest animal organs such as the intestine, heart, liver, and kidney to obtain corresponding primary cells. Compared with the kit using trypsin, the activity of the heat-resistant metalloprotease is not affected by serum, which is more suitable for scenarios where serum needs to be added during the digestion process.

Components

Components

S

M

L

Recombinant collagenase mixed components

1.0ml×2

4.0ml

10.0ml

Thermoresistant metalloproteinases

1.0ml×6

12.0ml

30.0ml


Size

Collagenase activity

Thermostable metalloproteinase activity

S

>3500CDU

>600U

M

>7000CDU

>1200U

L

>17500CDU

>3000U



Protocol

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

  • Preparation of digestive solution: 0.5ml recombinant collagenase mixed components + 1.5ml heat-resistant metalloproteinase, dilute with 4.0ml DMEM medium, and add FBS to a final concentration of 3%, DNase I To a final concentration of 100U/ml, and the blue chain double antibody to a final concentration of 0.1%;
  • Remove the small intestine from the tissue preservation solution, transfer it to a 10cm dish, add 10ml pre-cooled PBS + 1% blue chain double antibody, and rinse it 2-3 times;
  • Transfer the rinsed small intestine to a new 10cm dish containing 10ml pre-cooled PBS + 1% blue chain double antibody, rinse it back and forth 3 times with a syringe to remove the contents of the small intestine;
  • Transfer the rinsed small intestine to a new 10cm dish containing 10ml pre-cooled PBS + 1% blue chain double antibody, cut the small intestine longitudinally with scissors, and gently scrape off the villi on the surface of the small intestine with tweezers;
  • Transfer the small intestine to a new 10cm dish containing 10ml pre-cooled PBS + 1% blue chain double antibody, cut the small intestine into 1cm long segments, and transfer it to 2ml EP tube, cut into pieces with scissors appropriately;
  • Transfer the cut small intestine to a 15ml centrifuge tube, add 6ml digestion solution, and place in a 37℃ water bath for 10min. Take it out after 5min, mix it upside down and let it stand for a while, take the supernatant to a 50ml centrifuge tube, and add DMEM to complete the culture to 20ml; l
  • Add 5ml DMEM+3% FBS to the precipitate, blow it several times, let it stand for a while, take the supernatant and combine it with the solution in the previous step; l
  • Centrifuge at 340g and 4℃ for 8min, discard the supernatant, resuspend it with 12.5ml PBS and pass it through a 70µm cell sieve, and add 8ml DMEM complete culture medium to the sieved cells.

Guidelines

This product should avoid repeated freezing and thawing. If there is any surplus, it is recommended to store it at -20℃

Unit Definition

Collagenase activity definition: Within 5 hours, at pH 7.4, 37 °C and in the presence of calcium ions, the amount of enzyme that releases peptides from bovine Achilles tendon collagen and the ninhydrin color is equivalent to 1.0 µmol of leucine is 1 CDU. Thermostable metalloproteinase activity definition: at pH 7.5 and 37 °C, the amount of enzyme required to hydrolyze casein and produce 1.0 μM (181 μg) tyrosine per minute is 1U.
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SKU: 6609012859

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