The protocol used for myocyte isolation is described in detail elsewhere18 (link). Briefly, animals were anesthetized with pentobarbital, and hearts rapidly removed via thoracotomy. The aorta was cannulated on a Langendorff apparatus fitted with a heating jacket circulating water at 37°C, and retrograde-perfused for 5 minutes at 8 ml/min with Tyrode’s solution. The perfusate was then switched to Tyrode’s solution containing collagen type 2 (Worthington) and protease type 14 (Sigma-Aldrich) for 7 minutes. The solution was switched to a modified Kraft-Bruhe (KB) buffer for 5 minutes. The left ventricle was minced and filtered (200μm) to yield single cells. Myocytes rested in KB buffer19 (link) for an hour before being placed in supplemented M199 ACCIT medium20 (link).
Collagen type 2
Manufactured by Worthington
Collagen type II is a structural protein found in the extracellular matrix of cartilage. It is the primary component of articular cartilage and provides tensile strength and resilience to the tissue.
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Lab products found in correlation
5 protocols using collagen type 2
1
Isolation of Cardiac Myocytes
2
Guinea Pig Myocyte Isolation Protocol
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The protocol used for guinea pig myocyte isolation is described in detail elsewhere 19 .
Briefly, guinea pigs were anesthetized with pentobarbital, and hearts rapidly removed via thoracotomy. The aorta was cannulated on a Langendorff apparatus fitted with a heating jacket circulating water at 37 °C, and retrograde-perfused for 5 minutes at 8 ml/min with Tyrode's solution. The perfusate was then switched to Tyrode's solution containing collagen type 2 (Worthington, Columbus, OH) and protease type 14 (Sigma-Aldrich, St. Louis, MO) for 7-9 minutes. The solution was switched to a modified Kraft-Bruhe (KB) buffer for 5 minutes, before hearts were minced and filtered (200 µm) to yield single cells. Myocytes were then rested in modified KB buffer 20 for one hour before being placed in supplemented M199 ACCIT medium 21 . Cells were maintained at room temperature and studied over 7 hours.
Briefly, guinea pigs were anesthetized with pentobarbital, and hearts rapidly removed via thoracotomy. The aorta was cannulated on a Langendorff apparatus fitted with a heating jacket circulating water at 37 °C, and retrograde-perfused for 5 minutes at 8 ml/min with Tyrode's solution. The perfusate was then switched to Tyrode's solution containing collagen type 2 (Worthington, Columbus, OH) and protease type 14 (Sigma-Aldrich, St. Louis, MO) for 7-9 minutes. The solution was switched to a modified Kraft-Bruhe (KB) buffer for 5 minutes, before hearts were minced and filtered (200 µm) to yield single cells. Myocytes were then rested in modified KB buffer 20 for one hour before being placed in supplemented M199 ACCIT medium 21 . Cells were maintained at room temperature and studied over 7 hours.
3
Isolation of Adipose-Derived Stromal Cells
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Adipose tissue from interscapular or inguinal region were excised from mice for brown or white SVF, minced, and digested with 0.2% collagen type II (Worthington Biochemical) in SVF primary cells isolation buffer (100 mM HEPES, pH 7.4, 123 mM NaCl, 5 mM KCl, 1.3 mM CaCl2, 5 mM glucose, 1× penicillin/streptomycin, and 4% fraction V bovine serum albumin (Sigma)) for 30 min in shaking incubator set at 37 °C/150 rpm. During enzyme digestion in the shaking incubator, tubes were vortexed for 10 seconds every 5 min. After enzyme digestion, tubes with enzyme-digested solution were put on ice for 30 min to allow the mature adipocytes and lipid droplets to float. Clear part of enzyme-digested solution filtered through 70 µm cell strainer (BD Bioscience) and centrifuged at 700 × g for 10 min to pulldown SVF primary cells. After washing cell pellet with DMEM, SVF primary cell pellet was resuspended with prewarmed growth medium (DMEM with high glucose, 20% fetal bovine serum (FBS), and antibiotics) and plated on the cell culture dishes.
4
Isolation and Culture of Neonatal Rat Ventricular Myocytes
5
Isolation and Culture of Neonatal Rat Ventricular Cardiomyocytes
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Briefly, hearts were excised from 2 to 4 day old Sprague-Dawley rats, ventricles separated and minced thoroughly before digestion with Collagen type II (Worthington) in Hanks buffered saline solution (HBSS) without Ca2+ or Mg2+. Following digestion, cells were collected by centrifugation into Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM glutamine and 2 μg/mL vitamin B12. Contaminating cells were removed by preplating cells onto tissue culture plastic for a minimum of 1 hr at 37°C. NRVMs were then plated onto either glass-bottom tissue culture plates or 12 well plates coated with 0.2% gelatin and cultured in DMEM (composition as above, with additional 10 μM cytosine arabinoside). 48 hr later, cells were transferred into media supplemented with 1% FBS. For transduction with adenovirus, 50 MOI of indicated adenovirus was added overnight upon transfer to 1% FBS containing media. For fluorescent adenovirus constructs, expression was confirmed the next day by epifluorescence microscopy.
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