{ "ProductData": { "ID": "3.5.3.3.11.8", "ProductType": "Others", "BrandName": "Liberase T-Flex, Research Grade", "ProductNameAddition": "0.2 μm filtered, lyophilizate", "RegulatoryDisclaimer1": "For life science research only. Not for use in diagnostic procedures.", "DisclaimerGroup1": "LSR", "RegulatoryDisclaimer2": "", "DisclaimerGroup2": null, "RegulatoryDisclaimer3": "", "SampleType": [ ], "LicenseDisclaimers": [ ], "RelatedLinks": "", "Clone": "", "ControlTissue": [ "" ], "ISOtypes": "", "Species": [ "" ], "StainLocalization": [ "" ], "ProductNameGlobal": "Liberase T-Flex Research Grade" }, "ProductImageDetails": { "ImagePath": "https://pim-media.roche.com/Images/Article_05989132001_im_en.png", "ImageType": "Image main" }, "Product2Taxonomy": { "Product2TaxonomyReferences": [ { "StructureSystemIdentifier": "OWP_Family", "StructureSystemName": "Product Families", "StructureNodeStatus": "valid", "NodeName": "Liberase", "NodeID": "510" }, { "StructureSystemIdentifier": "OWP_Product_Types", "StructureSystemName": "Product Types", "StructureNodeStatus": "valid", "NodeName": "Proteases", "NodeID": "70-044-00" }, { "StructureSystemIdentifier": "Product_Grouping", "StructureSystemName": "Product Grouping", "StructureNodeStatus": "valid", "NodeName": "Cell Isolation", "NodeID": "02-0134" }, { "StructureSystemIdentifier": "Product_Grouping", "StructureSystemName": "Product Grouping", "StructureNodeStatus": "valid", "NodeName": "Cell & Gene Therapy", "NodeID": "02-0133" }, { "StructureSystemIdentifier": "OWP_Organization", "StructureSystemName": "Organization", "StructureNodeStatus": "valid", "NodeName": "CustomBiotech and LifeScience", "NodeID": "01-01-01-07-00" }, { "StructureSystemIdentifier": "Product_Grouping", "StructureSystemName": "Product Grouping", "StructureNodeStatus": "valid", "NodeName": "Enzymes", "NodeID": "02-0014" }, { "StructureSystemIdentifier": "OWP_Techniques", "StructureSystemName": "Techniques", "StructureNodeStatus": "valid", "NodeName": "Tissue Dissociation", "NodeID": "330-00" }, { "StructureSystemIdentifier": "Product_Solutions", "StructureSystemName": "Product Solutions", "StructureNodeStatus": "valid", "NodeName": "CustomBiotech", "NodeID": "520" }, { "StructureSystemIdentifier": "Lab_Type", "StructureSystemName": "Lab Types", "StructureNodeStatus": "invalid", "NodeName": "", "NodeID": "180-00" }, { "StructureSystemIdentifier": "Applications", "StructureSystemName": "Applications", "StructureNodeStatus": "invalid", "NodeName": "", "NodeID": "99-00-00" }, { "StructureSystemIdentifier": "Disease_Areas", "StructureSystemName": "Disease Areas", "StructureNodeStatus": "invalid", "NodeName": "", "NodeID": "99-00-00" }, { "StructureSystemIdentifier": "Pathogens", "StructureSystemName": "Pathogens", "StructureNodeStatus": "invalid", "NodeName": "", "NodeID": "99-00-00" }, { "StructureSystemIdentifier": "Health_Topics", "StructureSystemName": "Health Topics", "StructureNodeStatus": "invalid", "NodeName": "", "NodeID": "99-00-00" }, { "StructureSystemIdentifier": "Product_Grouping", "StructureSystemName": "Product Grouping", "StructureNodeStatus": "valid", "NodeName": "Biologics", "NodeID": "02-0008" } ] }, "Product2Materials": { "P2MaterialReferences": [ { "MaterialNum": "05989132001", "MaterialDescription": "Liberase T-Flex Research Grade", "RegisteredProductName": "Liberase T-Flex, Research Grade", "GTIN": "07613336132081", "ProductCategoryText": "Reagents, kits", "OldMaterialNumber": "", "PackSizePIM360": "1 kit", "PackSizeDescPIM360": "1 × 500 mg Collagenase blend
2 × 15 mg Thermolysin", "MaterialAnnotation": "CustomBiotech product. Please contact your local representative.", "ReadyForUse": "", "OrderInformation": "Will be supplied as \"Liberase T-Flex Research Grade\". Unit of measure is \"piece\"." } ] }, "Product2Products": { "Product2ProductReference": [ ] }, "ProductSpec": [ { "ProductSpecVariant": { "Chapters": [ { "Language": "en", "Value": "The Liberase T-Flex Research Grade kit contains highly purified collagenase class I and class II from Clostridium histolyticum. The two collagenase isoforms are blended in a precise ratio with each other. Highly purified thermolysin from Bacillus thermoproteolyticus is provided in a separate vial to allow individual adjustments of enzyme ratios for optimization of your tissue dissociation protocol.", "Country": "XG", "Code": "Product Description", "Name": "Product Description" }, { "Language": "en", "Value": "Use Liberase T-Flex, Research Grade, for in vitro enzymatic dissociation of a broad range of tissues, enabling isolation of single cells. In particular intended for use in isolation procedures of pancreatic islets from several animal types.", "Country": "XG", "Code": "Applications", "Name": "Applications" }, { "Language": "en", "Value": "

• Protect respiratory system, eyes, and skin when handling proteases.
• Open the vials inside a laminar flow hood.

• Handle all samples as if potentially infectious, using safe laboratory procedures. As the sensitivity and titer of potential pathogens in the sample material varies, the operator must optimize pathogen inactivation by the Lysis / Binding Buffer or take appropriate measures, according to local safety regulations.
• Do not eat, drink or smoke in the laboratory work area.
• Do not pipette by mouth.
• Wear protective disposable gloves, laboratory coats and eye protection, when handling samples and kit reagents.
• Wash hands thoroughly after handling samples and reagents.

• Discard unused reagents and waste in accordance with country, federal, state, and local regulations.
• Safety Data Sheets (SDS) are available online on dialog.roche.com, or upon request from the local Roche office.

", "Country": "XG", "Code": "Safety Information", "Name": "Safety Information" }, { "Language": "en", "Value": "

Problem	Possible cause	Recommendation
Prolonged dissociation time or incomplete dissociation.	Enzyme decay.	Follow appropriate storage conditions, see section, Storage Conditions (Product) and Storage Conditions (Working Solution).
Inappropriate Enzyme reconstitution time.	Follow appropriate reconstitution conditions, see section, Reconstitution.
Inappropriate Enzyme dilution.	Verify dilution.
Enzyme inhibition or tissue exposed to enzyme inhibitors.	Check for presence of inhibitors in all buffers, see section, Inhibition.
Incubation temperature too low.	Verify +37°C is incubation temperature.
Low cell viability and yield.	Tissue stored at elevated temperature prior to dissociation.	Reduce time and temperature of ischemia.
Prolonged tissue ischemia time.	Reduce time of tissue ischemia.
Incubation time too long.	Reduce incubation time.
Inappropriate Research Grade dilution.	Verify dilution.
Incubation temperature too high.	Verify +37°C is incubation temperature.
Decreased cell viability or in vitro survival.	Endotoxin exposure.	Check all tissue dissociation reagents for endotoxin contamination.
Liberase Research Grade does not go into solution within 30 minutes.	The volume used for the reconstitution of the lyophilized enzyme is too low.	Increase volume of reconstitution buffer two-fold.

", "Country": "XG", "Code": "Troubleshooting", "Name": "Troubleshooting" }, { "Language": "en", "Value": "Will be supplied as \"Liberase T-Flex Research Grade\". Unit of measure is \"piece\".", "Country": "XG", "Code": "CB - Order Information", "Name": "CB - Order Information" }, { "Language": "en", "Value": "

• Tissue dissociation buffer, or
• Phosphate buffered buffer (PBS)

", "Country": "XG", "Code": "Additional Equipment and Reagent Required", "Name": "Additional Equipment and Reagent Required" }, { "Language": "en", "Value": "

Vial / Bottle	Cap	Label	Function / Description	Content
1	red	Collagenase I/II Blend Research Grade (500 mg)	White lyophilizate. Enzyme blend Collagenase I/II and a small quantity of buffer salts.	1 bottle, 500 mg
2	red	Thermolysin Research Grade (15 mg)	White lyophilizate. Enzyme Thermolysin and a small quantity of buffer salts.	2 bottles, 15 mg each

", "Country": "XG", "Code": "Content", "Name": "Content" }, { "Language": "en", "Value": "Target Activities
Each vial of Liberase T-Flex Research Grade purified enzyme is filled by total protein mass. Combined collagenolytic activity of the Collagenase I and II isoforms is measured by the method of Wünsch (1). Neutral protease activity is measured by a non-fluorescent Caseine assay (2). The kit components allow to make individual blends of Collagenase and Thermolysin ratios.
Composition of Liberase T-Flex Research Grade Purified Enzyme Blends

Product	Target Enzyme Mix [mg]	Target Collagenase Activity [Wünsch U/vial]	Target Neutral ProteaseAmount	Enzyme Mixture Aggressiveness(1)	Main Application
Collagenase I/II Blend Research Grade Thermolysin Research Grade	500	2600	Low	+(+)	Very gentle isolation of islets from pancreas
15
Collagenase I/II Blend Research Grade Thermolysin Research Grade	500	2600	Low	++	Isolation of islets from rodent pancreas
24
Collagenase I/II Blend Research Grade Thermolysin Research Grade	500	2600	Low	++(+)	More harsh isolation of islets from pancreas
30

1. + equals lowest neutral protease activity/mg protein and +++++ equals highest neutral protease activity/mg protein.

This section will help you interpret your tissue dissociation results, and find opportunities to improve your cell yield, viability, and/or functionality. Before continuing, see section, Protocols, Factors affecting Liberase Research Grade selection regarding enzyme requirements for tissue dissociation, as well as the points below:

• Liberase T-Flex Research Grade Purified Enzyme Blends contain only collagenase and neutral protease.
• Collagenase enzymes digest the intercellular matrix.
• Neutral proteases act synergistic with collagenase.
• Given sufficient time and concentration, neutral proteases damage cell surface proteins.
• Time of dissociation, enzyme ratios, and enzyme concentration all affect the tissue-dissociation outcome.
• Use Liberase T-Flex Research Grade without modifying factors, such as serum, BSA, or protease inhibitors.

 Use the following table for optimization in the sequence provided.

1. Note whether the yield, viability, or functionality of your cells isolated with Liberase Research Grade Purified Enzyme Blend is less than optimal.
2. Find the probable cause, then act on the recommendation.
3. Refer to enzyme mixture aggressiveness described in section, Target activities for information on neutral protease specific activity increasing within the Liberase Research Grade panel.

Observation 1	Observation 2	Possible Cause	Recommendation
Low cell viability.	Dissociation very rapid.	Enzyme concentration too high.	Reduce enzyme concentration by 50%.
Enzyme mixture aggressiveness too high.	Select a Liberase Research Grade Purified Enzyme Blend containing lower amounts of neutral protease.
Dissociation very slow.	Enzyme concentration too low.	Increase enzyme concentration by 50%.
Enzyme mixture aggressiveness too low.	Select a Liberase Research Grade Purified Enzyme Blend containing higher amounts of neutral protease.
Impaired cell function.	Cell viability >80%; cell yield is reasonable.	Enzyme concentration too high.	Reduce enzyme concentration by 25%.
Enzyme mixture aggressiveness too high.	Select a Liberase Research Grade Purified Enzyme Blend containing lower amounts of neutral protease.
Low cell yield.	Cell viability >80%.	Enzyme concentration too low.	Increase enzyme concentration by 25 to 50%.
Enzyme mixture aggressiveness too low.	Select a Liberase Research Grade Purified Enzyme Blend containing higher amounts of neutral protease.
Cell viability <80%.	Enzyme concentration too high.	Reduce enzyme concentration by 50%.
Enzyme mixture aggressiveness too high.	Select a Liberase Research Grade Purified Enzyme Blend containing lower amounts of neutral protease.
Mechanical (shear) force is excessive	Reduce shear force in all aspects of dissociation. Treat tissue gently.
Released cells clump in gelatinous stringy form.	Cell yield and viability are acceptable	DNA release subsequent to cell lysis, causes clumping.	More prevalent in some tissues. If cell viability is acceptable, add DNase to dissociation mixture.
Cell yield or viability are reduced.	Mechanical (shear) force is excessive.	Reduce shear force in all aspects of dissociation. Treat tissue gently.

", "Country": "XG", "Code": "General Considerations", "Name": "General Considerations" }, { "Language": "en", "Value": "Liberase T-Flex Research Grade kit contains enzymes of purified collagenase isoforms I and II, and the neutral protease Thermolysin. The collagenase isoforms are purified from the fermentation of Clostridium histolyticum. Thermolysin is purified from the fermentation of Bacillus thermoproteolyticus. The methods for purification and blending of these components are described in one or more of the following patent numbers: US 5,753,485 and US 5,830,741.", "Country": "XG", "Code": "Principle", "Name": "Principle" }, { "Language": "en", "Value": "The Liberase T-Flex Research Grade kit enables to make individual blends of collagenase and Thermolysin ratios. As a guideline please refer to the ratios described in the section “Target Activities” (Section General Considerations). Mix different ratio of neutral protease activity, relative to the collagenase activity to get an enzyme blend appropriate for your application.", "Country": "XG", "Code": "Applications (IFU)", "Name": "Applications (IFU)" }, { "Language": "en", "Value": "Prior to choosing a Liberase Research Grade for your application, familiarize yourself with the factors that influence enzyme requirements. Enzyme requirements for tissue dissociation are determined by:

• Tissue type
• Species
• Dissociation protocol
• Desired outcome of tissue dissociation

", "Country": "XG", "Code": "Protocols", "Name": "Protocols" }, { "Language": "en", "Value": "The Liberase T-Flex, Research Grade kit enables individual blends of collagenase and thermolysin in low level ratios. The enzymes are manufactured for applications in research where high purity, consistent quality and low levels of bacterial endotoxins are required for reproducible high cell yield and viability.", "Country": "XG", "Code": "Positioning", "Name": "Positioning" }, { "Language": "en", "Value": "I. Collagenase I/II MPB
Appearance: White lyophilized cake
Protein mass/bottle (target: 537 mg protein): 430-644 mg protein
Collagenase I content(target: 322 mg): 258-386 mg
Collagenase II content(target: 215 mg): 172-258 mg
Collagenase Ib, c part of Collagenase I: ≤10 area%
HPLC purity(total peak areas Collagenase I+II): ≥85 area%
Activity (Wünsch), calculated: 2,172-3,617 U/bottle
Ratio Collagenase II/total Collagenase: 0.3-0.5
Endotoxins: ≤10 EU/mg protein
Bioburden: ≤10 CFU/bottle

II. Thermolysin MPB
Appearance: White lyophilized cake
Protein mass/bottle (target: 15.0 mg protein): 12.0-18.0 mg protein
Activity target neutral protease (calculated): 130,500-234,000 U/bottle
HPLC purity (total peak area thermolysin): ≥85 area%
Endotoxins: ≤50 EU/mg protein
Bioburden: ≤10 CFU/bottle", "Country": "XG", "Code": "Specification", "Name": "Specification" }, { "Language": "en", "Value": "CAS Number: 9001-12-1 and 9073-78-3", "Country": "XG", "Code": "Properties", "Name": "Properties" } ] } } ] }