Aortic tissue samples were placed into Waymouth's Medium for Aortic Biopsy supplemented with 3% Sodium Bicarbonate, 1.25% 200 mmol/L l ‐Glutamine (100×), 1% Antibiotic‐Antimycotic (Anti‐Anti) (100×), 1% 1 mol/L 4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid (HEPES) (1×) and 1% Minimum Essential Medium Non‐Essential Amino Acid Solution (MEM NEAA) (100×) (ThermoFisher Scientific) and transferred to a cell culture hood. Intima and adventitia layers were discarded, while the tunica media layer was placed into a culture dish containing 5 mL of aortic biopsy media with 0.1 mg/mL Collagenase Type I and 0.025 mg/mL Soybean Trypsin Inhibitor. The aortic tissue was minced and the dish placed in an incubator overnight at 37° C and 5% CO2. Foetal bovine serum (Lonza) was added after incubation to arrest digestion. The contents were collected and centrifuged; the supernatant was removed and the pellet was released with 4 mL of SMC culture media that consisted of Smooth Muscle Basal Media (Lonza), 10% FBS, SmGm‐2 SingleQuot kit (Insulin, rhFGF, rhEGF) (#CC3182 Lonza), 1% Anti‐Anti 100×, 1% 200 mmol/L l ‐Glutamine (100×), 1% 100 mmol/L Sodium Pyruvate (100×), and 2% 1 mol/L HEPES (1×). Cell lines were maintained in SMC media and passaged once culture flasks reached 70%‐80% confluency.
Smgm 2 singlequot kit
Manufactured by Lonza
The SmGM-2 SingleQuot kit is a laboratory product provided by Lonza. It contains a set of supplements and growth factors that are designed to support the culture and growth of smooth muscle cells in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Smooth muscle growth medium 2, by Lonza (3 mentions)
Mem neaa, by Thermo Fisher Scientific (1 mentions)
Smooth muscle basal media, by Lonza (1 mentions)
Foetal bovine serum (fbs), by Lonza (1 mentions)
Rasmcs, by Lonza (1 mentions)
Cholesterol mβcd, by Merck Group (1 mentions)
Pd123319, by Merck Group (1 mentions)
Parthenolide, by Merck Group (1 mentions)
Irbesartan, by Merck Group (1 mentions)
Tgf β, by R&D Systems (1 mentions)
Angii, by Bachem (1 mentions)
5 protocols using smgm 2 singlequot kit
1
Aortic Smooth Muscle Cell Isolation
2
Rat Aortic Smooth Muscle Cell Culture
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Rat aortic smooth muscle cells (RASMCs; Lonza) were cultured as previously described (Rahaman et al., 2017 (link)). In brief, RASMCs were cultured at 37°C in Lonza Smooth Muscle Growth Medium-2 with SmGm-2 SingleQuot kit (Lonza) supplementation. Cells were passaged using Gibco Trypsin/EDTA up to passage 12. For treatment, FoxO inhibitor AS1842856 (Cayman) was dissolved in DMSO at 10 mM stock concentration and diluted in DMSO for further experiments. Control and treatment DMSO concentration was 0.1% of the total volume for treatment periods.
3
Murine Aortic Contractility Assay
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The following treatment method was performed as previously described (Rahaman et al., 2015 (link)). In brief, isolated murine thoracic aortas were isolated from mice and incubated with 10 µM FoxO inhibitor for 48 hours in one part Lonza Smooth Muscle Growth Medium-2 supplemented with SmGM-2 SingleQuot kit to nine parts unsupplemented Lonza Smooth Muscle Growth Medium-2. Following treatment, aortas were cut into 2-mm rings before being placed on a two-pin myograph (Danish Myo Technology). Aortic rings were then incubated in a physiologic salt solution (PSS) for 30 minutes of rest, after which 500-mg tension was applied to the vessels. Vessel viability was tested using potassium physiologic salt solution for 15 minutes, followed by a triplicate of PSS washes and a 60-minute resting period. Cumulative concentration responses to phenylephrine (10−9–10−5) and sodium nitroprusside (SNP; 10−9–10−4) determined vessel contractility and relaxation responses, respectively. Finally, relaxation percentage was determined by normalizing cumulative SNP relaxation to maximal contraction at 10−5 M phenylephrine and maximal dilation at 10−4 M SNP in Ca2+-free PSS.
4
Rat Aortic Smooth Muscle Cell Culture
5
Cultured Coronary Artery Smooth Muscle Cells
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Human coronary artery smooth muscle cells (caHSMCs, Lonza Cat #CC-2583) were cultured in SmBM media (Lonza, Cat #CC-3181) with 5% fetal bovine serum and SmGM-2 SingleQuot Kit (Lonza, CC-4149) and maintained at 37 °C in a humidified atmosphere containing 5% CO2. Cells were used between passage 3–7 and at 80–90% confluency before treatment. caHSMCs were serum starved for 12 h and treated with Mβ-CD Cholesterol (10 µγ/ul, Sigma, Cat. #C4951) LPA (1 µM), AngII (1 µM, Bachem, Cat. # 4006473), transforming growth factor beta 1 (TGFβ) (1 µM, R&D Systems, Cat. #240-B002), parthenolide (1 µM, Sigma Cat. #P0667), Irbesartan (Sigma, Cat. #61188) or PD-123-319 (Sigma, Cat. #P186) for either: 24, 48 or 72 h.
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