For breast tissue enzymatic disaggregation what do you prefer Trypsin or collagenase and what are precautions I should take in choosing the media and other reagents for every one of them.
Thanks
Hi Mohamed , Breast Tissue Dissociation System, OptiTDS™, have the enzyme components: Collagenase I: from Clostridium Histolyticum, Collagenase III: from Clostridium Histolyticum, Collagenase IV: from Clostridium Histolyticum,Collagenase: from Clostridium Histolyticum, Trypsin: from Bovine Pancreas obtained from CHI scientific with Breast OptiTDS™ Digestion Buffer and following this procedure.
1. Prepare fresh enzyme working solutions: add 1.0 ml of Breast Tissue Dissociation System, OptiTDS™, to one vial of Breast OptiTDS™ Digestion Buffer (9.0 ml). Warm the diluted enzyme working solution at 37 ℃ for 10 min prior to use. For optimal results of performance, we recommend use 4-5 g tissue samples per 10 ml diluted enzyme working solutions.
2. Mince pre-washed tissue into pieces approximately 0.2-0.5 mm2 in diameter with two scalpel and forceps.
3. Incubate minced tissues with the enzyme working solutions by incubating minced tissue samples (up to 5 g) in 10 ml fresh enzyme working solutions with slow magnetic stirring for 30 min at 37 ℃.
4. At the end of tissue dissociation period, gentle triturating tissue with a 10 ml pipette, constitutes filling the empty the barrel at a rate of 2-3 ml per second. Repeat this procedure for 5-6 times.
5. Collecting cells by filtration the mixture through a cell strainer to remove fatty layer and larger undigested tissues followed by centrifugation at 1 x 100 g. carefully collect enzyme working solutions and resuspend the cell pellet with 1.0 ml complete culture medium.
6. Count the cells and seed cells in 3-4 T-25 collagen I-coated flasks (Important: pre-treat the flask with the provided Coating Solutions for 5 min and air-dry in hood prior to use) upon the density of viable cells (2.5-5 x 105 Cells/flask)
regards
Trypsin is use to dis-aggregate musculat tissue mainly contain proteoglycans or lipoproteins or any polymer or conjugant of two biomolecules. Collagenase is used to extract out enzymes from squamous epithelial and fibrous tissues or from malignant cells .
Affymetrix Cancer Cell Isolation kit gives good results. The kit can be used for isolating cells from different tissue origins.
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