Neonatal Cardiomyocyte Isolation System
The Worthington Neonatal Cardiomyocyte Isolation System has been developed to provide researchers with a reliable, convenient, and consistent neonatal rat cardiomyocyte cell isolation method. By using purified rather than crude enzyme preparations, it has been possible to minimize the lot-to-lot variation. In addition, Worthington use-tests the kits by isolating cardiomyocytes from neonatal rat hearts to assure performance, reliability and consistent yield of viable cells.
The kit has been formulated in collaboration with Dr. Ronal MacGregor. The method is based on that described by Toraason, et al. Toxicol, 56, 107 (1988) in which the minced tissue is incubated overnight with purified trypsin at 2-8°C. As pointed out by Toraason, this step reduces the hands-on time required to harvest cells compared to the time involved in sequential incubations in warm trypsin or collagenase. Purified collagenase rather than crude collagenase is used to maximize yield and viability.
Contents of Kit
The package contains sufficient materials for five separate tissue dissociations, each containing up to twelve hearts. For larger or smaller tissue samples prepare proportionate volumes of reagents at each step and combine them in the same ratio as described in the protocol.
• Vial 1: 1 bottle, 500ml: Sterile calcium- and magnesium-free Hank's Balanced Salt Solution (CMF HBSS), pH 7.4. The solution is used for reconstituting the contents of Vials #2 and #3 in addition to serving as the medium for the dissociation.
• Vial 2: 5 vials, 1000µg each: Worthington Trypsin (Code: TRLS), 3X crystallized, dialyzed against 1mM HCl, filtered through 0.22 micron pore size membrane, and lyophilized. Before use, reconstitute with 2ml CMF HBSS (Vial #1) and swirl gently to dissolve contents. Store at 2-8°C.
• Vial 3: 5 vials, 2000µg each: Worthington Soybean Trypsin Inhibitor (Code: SIC), a 0.22 micron pore size membrane filtered, lyophilized powder. Before use, reconstitute with 1ml CMF HBSS (Vial #1) and swirl gently to dissolve contents. Store at 2-8°C.
• Vial 4: 5 vials, 1500 Units each: Worthington Purified Collagenase (Code: CLSPA), a 0.22 micron pore size membrane filtered, lyophilized powder which has been chromatographically purified. It contains less than 50 caseinase units per milligram and is composed of two separable but very similar collagenases. Before use, reconstitute with 5ml Leibovitz L-15 media (prepared as described below) and swirl gently to dissolve contents. Store at 2-8°C.
• Pouch containing Leibovitz L-15 Media Powder: 1 x 1L, Reconstitute entire contents of pouch by cutting open top of envelope and pouring contents into beaker containing 800ml of cell culture grade water. Rinse pouch 2-3 times with additional 100ml. Bring total volume to 1 liter and filter through a 0.22 micron pore size filter.
The kit also includes 5 Cell Strainers (Falcon), a card correlating phenol red color with pH for checking balanced salt solutions and culture media.
Deoxyribonuclease I (DP/D/DCLS/D2/DPFF/DPRF)
Neutral Protease (Dispase, NPRO)
Proteinase K (PROK)
Trypsin (TL/TRL/TRL3/TRLS/TRTPCK)Trypsin Inhibitors (LBI/OI/SI/SIC)
Cell Isolation Optimizimg System (CIT)
Hepatocyte Isolation System (HIS)
Papain Dissociiation System (PDS/PDS2)