Transcript papaya
Enzymatic disaggregation Tissue Dissociation Enzyme Specificities many enzyme systems have been applied for a particular type of cell isolation:- Collagenase : • • • most commonly is derived from Clostridium histolyticum degrades the helical regions in native collagen most commonly used for tissue dissociation collagenase most commonly used for tissue dissociation is a crude preparation containing clostridiopeptidase A in addition to a number of other proteases, polysaccharidases and lipases. Collagenases Subtypes Type 1 Containing average amounts of assayed activities (collagenase, caseinase, clostripain, and tryptic activities). It is generally recommended for epithelial, liver, lung, fat, and adrenal tissue cell preparations. Type 2 Containing greater clostripain activity. It is generally used for heart, bone, muscle, thyroid and cartilage. Type 3 Selected because of low proteolytic activity. It is usually used for mammary cells. Type 4 Selected because of low tryptic activity. It is commonly used for islets and other applications where receptor integrity is crucial. Trypsin pancreatic serine protease with specificity for peptide bonds involving the carboxyl group of the basic amino acids, arginine and lysine most highly specific proteases known alone is usually ineffective for tissue dissociation shows little selectivity for extracellular proteins Combinations of purified trypsin and other enzymes such as elastase and/or collagenase have proven effective for dissociation. Enzymes used for disaggregation – trypsin + EDTA or only trypsin Warm trypsin - @ 37°C + removed by centrifugation(1000 rpm for 10 to 15 min ) + neutralized with serum and medium + washing with PBS Cold trypsin – Soak tissue in trypsin at 4°C for 16 – 18 hrs. followed by incubation at 37°C for 20-30 minutes for disaggregation Differences between warm and cold trypsin treatment Warm trypsin Shorter time period Cold trypsin Gives higher yield of viable cells with improved survival after 24 h culture Centrifugation is required More effective but Lesser yield No stirring or centrifugation is required Usually used for the tissues containing extracellular matrix and fibrous connective tissues Incubation at 4 C can be done overnight More yield but less effective as compared to warm usually used for soft tissues like Embryonic organs etc Elastase serine protease with specificity for peptide bonds adjacent to neutral amino acids. also exhibits esterase and amidase activity. Unique in its ability to hydrolyze native elastin, a substrate not attacked by trypsin, chymotrypsin or pepsin. produced in the pancreas as an inactive zymogen, proelastase, and activated in the duodenum by trypsin. used with other enzymes like trypsin or collagenase to dissociate tissues which contain extensive intercellular fiber networks. Hyaluronidase is a polysaccharidase specifically cleavage of endo-Nacetylhexosaminic bonds between 2-acetoamido-2-deoxy-beta-D-glucose and D-glucuronate. hyaluronidaseis often used in combination with a crude protease such as collagenase, for the dissociation of connective tissues. Papain is a sulfhydryl protease from Carica papaya latex has wide specificity and degrades most protein substrates more extensively than the pancreatic proteases also exhibits esterase activity With some tissues has proved less damaging more effectiveidely used for obtaining high yields of viable, morphologically intact cortical neurons. Chymotrypsin preferentially catalyzes the hydrolysis of peptide bonds involving the aromatic amino acids tyrosine, phenylalanine, and tryptophan Chymotrypsin is used to a limited extent in tissue dissociation, usually in combination with trypsin and elastase. Neutral Protease (Dispase) is a bacterial enzyme produced by Bacillus polymyxa hydrolyses N-terminal peptide bonds of non-polar amino acid residues is classified as an amino-endopeptidase unique feature of this enzyme it possesses a mild proteolytic dissociation activity while preserving the integrity of the cell membrane. used as a secondary enzyme in conjunction with collagenase and/or other proteases in many primary cell isolation and tissue dissociation applications. dissociates fibroblast-like cells more efficiently than epithelial-like cells. Thus it has been used for differential isolation and culture applications.