1.1. Can the NCIS Kit be used on adults?
The NCIS kit is specifically packaged for five separate tissue dissociations, each containing up to twelve neonatal rat hearts. The NCIS protocol is available online at www.worthington-biochem.com/NCIS/default.html. We have a modified adult rat cardiomyocyte isolation tutorial that we can fax upon request which recommends the Langendorf Perfusion System and Collagenase Type II.1.2. What are the volumes of Hank's, Earle's and Leibovitz L-15 Media that you sell?
Sterile calcium and magnesium free Hank's Balanced Salt Solution, p 7.4 is supplied in the NCIS (Neonatal Cardiomyocyte Isolation System) kit. A 500ml bottle may be purchased, catalog #LK003210 is $42.The NCIS kit also contains a pouch of Leibovitz L-15 Media Powder. Reconstitute the entire contents of the pouch into a beaker containing 800ml of cell culture grade water. Rinse pouce 2-3 times with additional 100ml. Bring total volume to 1 liter and filter through a 0.22 micron pore size filter. The pouch containing Leibovitz L-15 Media powder for 1 liter, catalog #LK003250 is $18.
Sterile Earle's Balanced Salt Solution (EBSS) with calcium, magnesium, bicarbonate and phenol red is supplied with the PDS (Papain Dissociation System) kit. A 100 ml bottle may be purchased, catalog #LK003182 is $40.
2.1.1. What is an acceptable enzyme diluent for Collagenase (CLS)?
Collagenase may be directly dissolved in a balanced salt solution or buffer of choice. It should be sterile filtered through a 0.22um pore size membrane. Its optimum pH range is 7-9. Calcium is required. Most balanced salt solutions have enough Ca. Collagenase is inhibited by metal chelating agents like cysteine, EDTA or o-phenanthroline. For our assay we dissolve in 0.05M TES with 0.36mM Calcium Chloride pH 7.52.1.2. What is an acceptable enzyme diluent for Dispase (DISP)?
Dispase can be dissolved in water, balanced salt solution or buffer of choice. Calcium is an activator similar to collagenase.2.1.3. What are acceptable enzyme diluents for Deoxyribonuclease (D/DPFF/DPRF)?
D/DPFF: May be dissolved in 5mM acetate, 1 mM calcium, pH 4.5 and stored in single use aliquots at -20 or -70 for 1 yr.DPRF: Use water or any buffer pH 4-9 except phosphate, avoid calcium chelators, add 50% glycerol for storage as liquid at -20C. Aliquot avoid repeated freeze/thaw cycles.
Special care must be taken with deoxyribonuclease. Mix gently to avoid shear denaturation. In general, Dnase I is activated by bivalent metals. Maximum activation is attained with Mg+ and Ca+. Dnase I is inhibited by EDTA and SDS. The optimum pH of Deoxyribonuclease I from Bovine Pancreas is 7.8.
2.1.4. What is an acceptable enzyme diluent for Elastase (ES)?
Elastase has poor solubility at neutral pH and concentrations greater than 0.25%. It is helpful to make primary solutions in KCl or alkaline buffers before diluting into reaction mixtures or media, compensating for ionic strength or pH changes. Elastase is stable at pH 4.0-10.4, optimum pH 8.5. Elastase is unstable at pH below 3.5. Elastase should be sterile filtered through a 0/.22um pore size membrane before use. DO NOT FREEZE.2.1.5. What are acceptable enzyme diluents for Papain (PAP/PAP2)?
The enzyme should be fully activated before use in a solution containing 1.1mM EDTA, 0.067mM mercaptoethanol and 5.5mM cysteine-HCL for 30 minutes. Papain is activated by cysteine, sulfide and sulfite. Stabilizing agents are EDTA, cysteine and dimercaptoethanol. Do not freeze aqueous suspensions. Optimum pH range is 6.0-7.0.2.1.6. What is an acceptable enzyme diluent for Trypsin (TRL)?
Stock solutions of trypsin should be made initially by reconstituting in 0.001N HCL. This solution ca be diluted in the digestion medium or buffer immediately prior to use. The optimum pH of Trypsin is approximately 8.0.2.2.1. What are conversion factors for units of Collagenase (CLS)?
Unfortunately there is no consistent conversion unit between Wunsch and Mandl units.Worthington assay is based on modified Mandl unit approx. 0.15 Wunsch units equal 200 Mandl units.
2.2.2. What are conversion factors for units of Deoxyribonuclease (D/DP/DPRFF)?
Note: Kunitz units as reported by other suppliers can be 2-4 times higher than Kunitz units as measured at Worthington. At Worthington 0.005 Kunitz unit digest 1 ug of lambda DNA in 10 min at 37C in 50mM Tris, 1 mMMg++, pH 7.8 in a 50ul reaction. Correlation of digestion units with Kunitz units is different for other DNA and buffer systems. When protease is present, units can not be correlated.2.2.3. What are conversion factors for units of Trypsin (TRL)?
1 mg TRL> 180 TAME units >10,350 BAEE units >3,450 USP/NF units2.3. What is the enzyme digestion scale?
2.4.1. What are the basic differences among the four types of collagenase?
| CLS I | Contains average amounts of assayed activities (collagenase, caseinase, tryptic and clostripain). Recommended for epithelial, liver (hepatocytes), lung, fat (adipocytes) and adrenal cell isolations. |
|---|---|
| CLS 2 | Contains greater proteolytic activities, especially clostripain activities. It is generally used for heart (cardiomyocytes), bone, muscle, thyroid, cartilage and endothelial cells. |
| CLS 3 | All activities lower than average. Low caseinase and low proteolytic activity. It is usually used for mammary cells and other soft tissues. |
| CLS 4 | Selected because of low tryptic activity. High collagenase required. It is commonly used for pancreatic islets and other applications where receptor integrity is crucial. |
2.4.2. What are the test specifications for each type of collagenase?
| collagenase | caseinase | clostripain | tryptic | A280 | |
|---|---|---|---|---|---|
| CLS 1 | ≥125 u/mg | ≥200 u/mg | ≥4.0 u/mg | ≥0.5 u/mg | report |
| CLS 2 | ≥125 u/mg | ≥200 u/mg | ≥3.5 u/mg | ≥0.1 u/mg | report |
| CLS 3 | ≥100 u/mg | ≥50 u/mg | ≥3.0 u/mg | ≥0.3 u/mg | report |
| CLS 4 | ≥160 u/mg | ≥100 u/mg | ≥3.0 u/mg | ≥0.1 u/mg | report |
| CLSPA | ≥500 u/mg | ≥50 u/mg | ≥2.0 u/mg | ≥0.25 u/mg | report |
