The bulbous arteriosus (BA) of teleost fish comprises vascular smooth muscle cells (VSMCs). By crossing with a previously reported Tg(tagln:egfp) smooth muscle cell transgenic reporter line (Liu et al. 2003 (link); Seiler et al. 2010 (link)) and adapting existing protocols for isolation of mammalian arterial VSM (Huang et al. 2018 (link)), we successfully isolated and identified ZF VSMCs from BAs (Fig. 1A ). BAs were excised from 4 ZF and placed in Solution 1 (S1; 0.1% BSA (w/v), 145 NaCl, 4 KCl, 10 HEPES, 10 glucose, 0.05 CaCl2, 1 MgCl2 (mM), adjusted to pH 7.4 using NaOH) on ice. S1 was then replaced by 400 μl Solution 2 (S2; 2 ml S1, 4 mg papain (Worthington), 2 mg DTT (Sigma)). The BA tissue was digested in S2 at 32°C on a shaking table (800 rpm) for 25 min. Following this, S2 was replaced by 500 μl Solution 3 (S3; 2 ml S1, 3 mg Collagenase Type H (Sigma), 2 mg trypsin inhibitor (Worthington), 1 mg elastase (Worthington)) and digested for additional 5 min. Digestion was terminated by replacing S3 with 500 μl S1. The tissue was triturated using Pasteur pipette and isolated cells were plated onto coverslips, then left to attach for at least 1 h before experiments and were used within 8 h.
Collagenase type h
Manufactured by Merck Group
Sourced in United States, Switzerland
Collagenase type H is a laboratory enzyme used for the digestion of collagen. It is a blend of proteolytic enzymes derived from Clostridium histolyticum that can effectively break down the collagen matrix in various tissues.
Automatically generated - may contain errors
Lab products found in correlation
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Trypsin inhibitor, by Worthington (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Papain, by Worthington (1 mentions)
Elastase, by Worthington (1 mentions)
Elastase, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Heat inactivated fetal bovine serum, by Thermo Fisher Scientific (1 mentions)
Sprague dawley rats, by BioLASCO (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Fungizone, by Merck Group (1 mentions)
6 protocols using collagenase type h
1
Isolation of Zebrafish Vascular Smooth Muscle Cells
2
Isolation of Intestinal Lymphocytes
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Intestinal biopsies were treated twice with 2 mM EDTA in PBS with 2% FCS at 37°C under constant rotation for 10 min in order to remove the epithelium and intraepithelial lymphocytes. The biopsies were subsequently digested with 1 mg/ml collagenase type H (Sigma) in 2% FCS in PBS at 37°C for 35 min under constant rotation. The digested tissue was homogenized with a syringe and needle and filtered through a 40-μm cell strainer and washed with PBS. Samples were then immediately used for analysis and/or sorting. 16 of the samples had been cryopreserved before flow cytometry analysis (Table S1 ).
3
Isolation of Coronary Artery Smooth Muscle Cells
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Hearts were excised and immediately transferred to ice-cold physiological saline solution containing (in mM): 134 NaCl, 6 KCl, 1 MgCl2, 2 CaCl2, 10 HEPES, and 7 glucose, pH 7.4. First and second-order branches of the left anterior descending coronary artery were manually dissected and smooth muscle cells were isolated as previously described27 . Briefly, arterial segments were incubated at 37 °C in Ca2+-free physiological saline (composition described above), containing papain (1 mg/ml) and dithiothreitol (1 mg/ml) for 5 min with gentle agitation. Then, the solution was replaced with buffer containing trypsin inhibitor (1 mg/ml) and collagenase (type H, 1 mg/ml) and incubated at 37 °C for an additional 5 min with gentle agitation. For human tissues, arterial myocytes were digested in collagenase (type H, 3 mg/mL; Sigma Aldrich), elastase (1 mg/mL; Sigma Aldrich), and bovine serum albumin (10 mg/mL; Sigma Aldrich) at 37 °C for 30 min with gentle agitation. Digested tissues were washed three times with ice-cold enzyme-free buffer. Cells were liberated by gentle trituration with a flame-polished glass pipette and kept on ice until use.
4
Osteoarthritis Chondrocyte Isolation Protocol
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Human chondrocytes were prepared as described in a previous study [38 (link)], and chondrocyte samples were obtained in accordance with protocols approved by the Institutional Review Board (IRB) of the Tri-Service General Hospital. Briefly, chondrocytes were harvested using articular cartilage from patients with osteoarthritis. For this, the cartilage was cut into 0.5 cm2 pieces using a sterile scalpel. Cartilage specimens then underwent digestion overnight using collagenase type H and 8× collagenase type II (Sigma, St. Louis, MD, USA, C-8015). Chondrocyte cells were collected using a cell strainer, seeded at a concentration of 58 × 105 cells in a 6 cm dish that contained dulbecco’s modified eagle medium (DMEM)/F12 medium with 10% FBS and antibiotics, and left to culture for 3 to 4 days prior to use.
5
Isolation and Culture of Rat Chondrocytes
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Male 3-week-old Sprague–Dawley rats (50–60 g each) were purchased from BioLASCO Taiwan (Taipei, Taiwan). The epiphyseal growth plate of the tibia was aseptically collected by cleaning the cartilage plate of muscular tissue, periosteum, and perichondrium. The proximal epiphysis was dissected with a sharp scalpel, and the cartilage plate was detached distally from the tibial metaphysis. Chondrocytes, which were obtained from tibial growth plates digested in 3 mg/mL of collagenase type H (Sigma)for 3 h at 37 °C, were cultured as monolayers in DMEM/F-12 medium, 10% heat-inactivated fetal bovine serum (GIBCO), 100 IU/mL penicillin (GIBCO), and 100 mg/mL streptomycin (GIBCO) in 5% CO2 and 95% air at 37 °C. Once fully confluent, the cells were harvested using trypsin-EDTA (GIBCO) and subcultured at a 1:3 ratio. The chondrocytes were routinely checked for positive anti-S100 protein (data not shown). A mycoplasma ELISA kit (Roche, Mannheim, Germany) was routinely used for thedetectionof contamination bymycoplasma in cell cultures.
6
Isolation and Culture of Human OA Chondrocytes
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Human cartilage and meniscus samples were obtained from surgical discard tissue, with consent (TMU-JIRB No.201305003), at knee joint arthroplasty from patients with OA (n = 43, mean age 72.86 years, range 56-84 years). Residual OA cartilage with predominantly grade II and III lesions (Collins/McElligot system) from each joint was removed and pooled. Cartilage and meniscus tissue were cut into small fragments, incubated with antimicrobial solution, containing 500 IU/mL penicillin (Gibco, Invitrogen, Burlington, Ontario, Canada), 500 mg/mL streptomycin (Gibco) and 2.5 μg/mL Fungizone (Sigma, St Louis, MO, USA) for 4 h, and then washed with sterile phosphate-buffered saline (PBS) before digestion. Cells were extracted by sequential enzymatic digestion with 0.25% trypsin (Gibco) and collagenase type H (Sigma).
Extracted cells were re-suspended in 10 mL Dulbecco's modified Eagle's medium/Nutrient Mixture F-12 HAM medium (Gibco) supplemented with 10% FBS (Gibco), 100 I.U./mL penicillin and 100 mg/mL streptomycin; seeded in complete medium at a density of 5 x 10 5 cell/mL in 60 mm Petri dishes (TPP, Trasadingen, Switzerland); and cultured in a humidified 5% CO 2 incubator at 37 o C for further experimental procedures. Cells between passages 3 and 5 were used.
Extracted cells were re-suspended in 10 mL Dulbecco's modified Eagle's medium/Nutrient Mixture F-12 HAM medium (Gibco) supplemented with 10% FBS (Gibco), 100 I.U./mL penicillin and 100 mg/mL streptomycin; seeded in complete medium at a density of 5 x 10 5 cell/mL in 60 mm Petri dishes (TPP, Trasadingen, Switzerland); and cultured in a humidified 5% CO 2 incubator at 37 o C for further experimental procedures. Cells between passages 3 and 5 were used.
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