Male Wistar rat (250–350 g) were anesthetized (Isoflurane, 2.5%–3%) and euthanized. Hearts were quickly excised, mounted on a Langendorff system, and perfused at 8 mL/min (37 °C) through the aorta with an oxygenated isolation solution containing (in mM): 130 NaCl, 5.4 KCl, 1.4 MgCl2, 0.4 NaH2PO4, 5 HEPES, 10 Glucose, 10 Creatine, 20 Taurine (pH 7.4 with NaOH), and 0.75 mM CaCl2. The hearts were then perfused with the isolation solution supplemented with 0.1 mM Ethyleneglycol- bis(β-aminoethyl)-N,N,N′,N′-tetraacetic Acid (EGTA) and finally with the same solution with the addition of collagenases (type II, 1 mg/mL; Worthington) and proteases (type XIV, 0.1 mg/mL; Sigma Aldrich) fwor 8 to 10 minutes. The left ventricles were then dissected, cut into small pieces and gently shaken at 37 °C in the enzymatic solution. Myocytes were collected by filtration through a nylon gauze and were used within 8 hours.45 (link)
Type 2
Manufactured by Worthington
Sourced in United States
Type II is a laboratory equipment designed for general use in scientific research and analysis. It serves as a versatile tool for various applications that require precise and controlled environmental conditions. The core function of Type II is to provide a controlled environment for experiments, sample preparation, or storage of sensitive materials. The specific details and intended use of this product are not included in this description.
Automatically generated - may contain errors
Lab products found in correlation
Collagenase, by Worthington (1 mentions)
Mem vitamin solution, by Thermo Fisher Scientific (1 mentions)
Ultra low adhesion polyhema coated 96 well plates, by Corning (1 mentions)
Protease type xiv, by Merck Group (1 mentions)
Collagenase type 1, by Worthington (1 mentions)
M199 medium, by Merck Group (1 mentions)
7 protocols using type 2
1
Isolation of Rat Ventricular Cardiomyocytes
2
Isolation of Mouse Ventricular Myocytes
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Adult mice were anesthetized with sodium pentobarbital (50 mg kg−1) and the excised heart was attached to an aortic cannula and perfused with solutions gassed with 100% O2 and held at 37 °C, pH 7.3. Perfusion with a 0 mM Ca2+ solution for 5 min was followed by 15 min of perfusion with the same solution containing 1 mg ml−1 collagenase (type II, Worthington Biochemical, Freehold, NJ, USA) and 0.1 mg/ml protease (type XIV, Sigma Chemical, catalog #P5147). The heart was then perfused for 1 min with a stopping solution (the same solution containing 20% serum and 0.2 mM CaCl2). All perfusions were performed at a flow rate of 2 ml min−1. The atria were removed and the ventricles were minced and shaken for 10 min, and then filtered through a nylon mesh. Cells were stored at 37 °C in a normal HEPES buffered solution. All myocytes used in this study were rod-shaped, had well-defined striations, and did not spontaneously contract. Experiments were performed within 7 h of isolation.
3
Isolation of Ventricular Myocytes from Mouse Hearts
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Myocytes were isolated from the LV of adult (10–20-wk-old) male and female WT, Fgf13 floxed (female Fgf13fl/fl and male Fgf13fl/y), cFgf13KO (female cFgf13−/− and male cFgf13−/y), and Fgf12KO mice by enzymatic and mechanical dissociation using previously described methods (Xu et al. 1999 (link); Brunet et al., 2004 (link)). In addition, LV myocytes were isolated from cFgf13KO mice (4–6 wk) after retro-orbital (eGFP- + FGF12B-expressing) AAV9 injections. Briefly, hearts were quickly removed from avertin-anesthetized mice and perfused retrogradely through the aorta with collagenase-containing solution (Type II, Worthington) at 37°C. After 15–20 min perfusion, the LV was separated, minced, and dispersed by gentle trituration. The resulting cell suspension was filtered and resuspended in serum-free medium-199 (M-199; Sigma-Aldrich). Isolated myocytes were plated on laminin-coated coverslips and maintained in a 95% air-5% CO2 incubator at 37°C until used (within 24–48 h) in electrophysiological experiments.
4
Bovine Primary Chondrocyte Isolation and Culture
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Primary chondrocytes were obtained by enzymatic digestion of minced fragments of femoral articular cartilage from juvenile bovine knee joints (2–6 months old, Research 87, MA), as previously described [27 (link),28 (link)]. After overnight collagenase digestion (Type II 298 U/mL Worthington, NJ, USA) isolated chondrocytes were filtered (100-μm and 70-μm and nylon meshes), washed with PBS, and cultured immediately for cartilage tissue analog fabrication. Cells were seeded in ultra-low adhesion polyHEMA coated 96-well plates (Corning, NY, USA) at 106 cells/well, allowing for tissue-like biomasses to form within 24 h of initial seeding. CTAs were maintained in complete media (high glucose DMEM) containing 10% FBS, 100U/mL penicillin, 100 μg/mL streptomycin, 2.5 μg/mL Fungizone [PSF, Life Technologies, NY, USA], 1% MEM Vitamin Solution [Gibco], 25 mM HEPES buffer, and 50 μg/mL ascorbic acid for at least 3 months (fresh media given twice a week) before being subject to compressive or chemical injury and treatment [28 (link)].
5
Isolation of Ventricular Myocytes from Pig Hearts
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Myocytes were isolated from the midmyocardial region of the left ventricle of explanted hearts of Yorkshire pigs following the procedures described in Klein et al.4 The left anterior descending coronary artery of explanted pig heart was perfused with low calcium Tyrode (in mmol/L: NaCl 145, KCl 4.5, MgCl2 1.6, NaH2PO4 0.33, Glucose 10, HEPES 10, EGTA 0.5, pH 7.4, 37 °C, oxygenated with 100% O2; 10 minutes), followed by Tyrode plus 0.75 mg/mL type II, 0.25 type I collagenase (Worthington) and 0.05 mg/mL type XIV protease (Sigma‐Aldrich; 30 minutes), then by Tyrode plus 0.1 mmol/L CaCl2 (10 minutes). Digested ventricular midwall was minced and strained through 200‐µm nylon mesh. Cells were stored in chilled Kraft‐Bruhe solution16 and used for electrophysiology within 36 hours.
6
Isolation of Atrial Myocytes from Rat Hearts
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Male rats (3-4 months of age) were killed by cervical dislocation according to the Home Office Guidance on the operation of the Animals (Scientific Procedures) Act 1986 (H.M.S.O.). The heart was rapidly excised, washed in a modified Tyrode medium containing EGTA and heparin and mounted on a Langendorff apparatus for retrograde perfusion via the aorta. Perfusion was initially carried out in a modified Tyrode solution containing (mM): NaCl 136, KCl 5.4, NaHCO3 12, Na + pyruvate 1, NaH2PO4 1, MgCl2 1, EGTA 0.04, glucose 5; gassed with 95% O2/5% CO2 to maintain a pH of 7.4. This was replaced after 2 minutes with a modified Tyrode solution containing 30 mg of collagenase and 10 μL of 0.1 mM CaCl2 (type II, Worthington Biochemical Corp., Lakewood, NJ, USA), but no EGTA. Additional 20 μL aliquots of 0.1 mM CaCl2 were added during perfusion. After this enzymatic digestion of the heart, atrial myocytes were isolated by trituration (mechanical disruption using a flamesmoothed glass pipette) and stored at 4 °C in KB medium containing (mM): KCl 70, MgCl2 5, K + glutamine 5, taurine 20, EGTA 0.04, succinic acid 5, KH2PO4 20, HEPES 5, glucose 10; pH to 7.2 with KOH.
7
Isolation and Culture of Cardiomyocytes
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Left ventricle cardiomyocytes were isolated as described elsewhere [22] . Briefly, hearts were retrogradely perfused (7.5 ml/min, 37 °C) with isolation buffer (130 mM NaCl, 1 mM L(+)-lactic acid, 5.4 mM KCl, 3 mM pyruvic acid, 25 mM HEPES, 0.5 mM MgCl 2 , 0.33 mM NaH 2 PO 4 , 22 mM Glucose and 50 μU/ml bovine insulin, pH = 7.4) containing 0.4 mM EGTA for 5 min, followed by 20 min with isolation buffer containing 0.048 mM CaCl 2 and 1 mg/ml collagenase (Type II, Worthington, USA). Left ventricule samples was minced and further digested by shaking (150 rpm, 37 °C) in isolation buffer containing 0.096 mM CaCl 2 and 20 mg/ml bovine serum albumin for 20 min. Cardiomyocytes were cultured in M199 medium (Sigma, USA) supplemented with 2 mg/ml bovine serum albumin, 1 μM insulin, 2 mM (±)-carnitine hydrochloride, 5 mM creatine, 5 mM taurine, 100 IU/ml penicillin and 100 μg/mg streptomycin.
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