Papain Dissociation System
The Worthington Papain Dissociation System is a set of reagents intended for use in the neural cell isolation method of Huettner and Baughman (J. Neurosci., 6, 3044, 1986). The materials are designed for convenience and simplicity and are useful to the occasional user as well as the more experienced and frequent user. Each lot is use tested for performance in rat spinal neural cell isolation and this kit provides freshly prepared enzyme solutions for each dissociation.
Stability/Storage: The reagents are stable at ambient temperatures for the periods of time expected in normal shipping procedures, but the package should be refrigerated upon arrival. Contents may be stored at 2-8°C for 4-6 months before use. Store at 2-8°C.
The package contains sufficient materials for dissociation of five separate tissue aliquots of up to 0.3-0.4 cm3 each. For larger tissue samples prepare proportionately larger volumes of reagents at each step and combine them in the same ratio as described in the protocol.
• Vial 1: Sterile Earle's Balanced Salt Solution (EBSS) with calcium, magnesium, bicarbonate and phenol red, one vial per package, 100ml. Aliquots of this vial are used to reconstitute other vials and to prepare dilute inhibitor solution. Refrigerate between uses and equilibrate with sterile O2:CO2 before each use.
• Vial 2: Papain containing L-cysteine and EDTA, 5 x 100 unit single-use vials per package. The material is 0.22 micron membrane filtered and lyophilized in autoclaved vials. A vial reconstituted with 5ml of EBSS (Vial 1) yields a solution at 20 units of papain per ml in 1mM L-cysteine with 0.5mM EDTA. Brief incubation at 37°C is needed to insure full solubility and activity.
• Vial 3: Deoxyribonuclease I (DNase), 5 x 2000 unit single use vials per package.
This material is 0.22 micron membrane filtered and lyophilized in autoclaved vials. A vial reconstituted with 0.5ml of EBSS (Vial 1) yields a solution at 2000 units of deoxyribonuclease per ml. Avoid vigorous mixing.
• Vial 4: Ovomucoid protease inhibitor with bovine serum albumin, one vial per package, 32ml upon reconstitution.
This material is 0.22 micron membrane filtered and lyophilized in autoclaved vials. A vial reconstituted with 32ml of EBSS (Vial 1) yields a solution at an effective concentration of 10mgs of ovomucoid inhibitor and 10mgs of albumin per ml. Aliquots of this vial are used for each dissociation. Refrigerate between uses and equilibrate with sterile O2:CO2 before each use. Stable after reconstitution when stored at 2-8°C.
Also included is a card correlating color with pH for use as a guide in O2:CO2 equilibration.
Deoxyribonuclease I (DP/D/DCLS/D2/DPFF/DPRF)
Neutral Protease (Dispase, NPRO)
Proteinase K (PROK)
Trypsin Inhibitors (LBI/OI/SI/SIC)
Cell Isolation Optimizimg System (CIT)
Hepatocyte Isolation System (HIS)
Neonatal Cardiomyocyte Isolation System (NCIS)