Vascular smooth muscle (VSM) cells were enzymatically isolated and cultured as previously described46 (link),47 (link). Briefly, F344XBN rat thoracic aortas were rinsed in Hanks balanced salt solution (HBSS) containing 50 μg/mL penicillin, 50 μg/mL streptomycin and 0.25 μg/mL amphotericin B (Gibco). After digestion for 30 min with 2 mg/mL collagenase I solution (Worthington Biomedical, Freehold, New Jersey) at 37 °C, the adventitia and intima layers were removed, and the vessel media was further digested with 2 mg/mL collagenase II/0.5 mg/mL elastase (Sigma) for 1 h at 37 °C. The isolated cells were washed and plated in complete medium. In all cases, >95% of cells stained positive for α-smooth muscle actin (α-SMA) and smooth muscle myosin heavy chains (SM1 and SM2) (Suppl. Fig. 1b,c ). Cells were maintained with VSMC medium including: DMEM (Gibco), 10% FBS (Sigma), 1% NEAA and 1% penicillin/streptomycin (Gibco). For all studies, we used early passage cells (passages 1–3).
Collagenase 1 solution
Manufactured by Worthington
Sourced in United States
Collagenase I solution is a laboratory reagent used to enzymatically digest collagen, a structural protein found in various tissues. It is commonly used in cell culture and tissue dissociation applications.
Automatically generated - may contain errors
Lab products found in correlation
Elastase, by Merck Group (3 mentions)
Collagenase 2, by Merck Group (3 mentions)
Amphotericin b, by Thermo Fisher Scientific (3 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Rhmfg e8, by R&D Systems (1 mentions)
Sb203580, by Santa Cruz Biotechnology (1 mentions)
3 protocols using collagenase 1 solution
1
Isolation and Culture of Vascular Smooth Muscle Cells
2
Isolation and Culture of Aged Rat VSMCs
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All in vitro experiments were performed utilizing rat VSMCs which were enzymatically isolated from aortae of rats aged 8mo (young) and 30mo (old), as previously described 10, 29 . Briefly, F344XBN rat thoracic aortae were rinsed in Hanks balanced salt solution (HBSS) containing 50μg/mL penicillin, 50μg/mL streptomycin and 0.25μg/mL amphotericin B (Thermo Fisher Scientific, Lanham, MD, USA). After digestion for 30 min in 2mg/mL of collagenase I solution (Worthington Biomedical, Freehold, NJ, USA) at 37°C, the adventitia and intima were removed from the vessel medial layer and then were further digested with 2mg/mL collagenase II/0.5mg/mL elastase (Sigma, Burlington, MA, USA) for 90-120 min at 37°C. Isolated VSMCs were washed and plated in complete medium containing 20% fetal bovine serum (FBS) and cultured at 37°C. Early passage (p3-p5) VSMCs were cultured in complete medium containing 10% FBS in the presence or absence of recombinant human MFG-E8 (rh-MFG-E8, R&D Systems, Inc., Minneapolis, MN, USA), and SB203580, a p38 inhibitor (Santa Cruz Biotechnology, Dalla, TX, USA).
3
Isolation and Culture of Rat Vascular Smooth Muscle Cells
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VSMCs were enzymatically isolated as previously described.5 , 6 , 19 , 20 Briefly, young and old AL and CR F344 rat thoracic aortae (n=3 animals/group) were rinsed in Hanks balanced salt solution containing 50 μg/mL penicillin, 50 μg/mL streptomycin, and 0.25 μg/mL amphotericin B (ThermoFisher). After digestion for 30 minutes in 2 mg/mL collagenase I solution (Worthington Biomedical, Freehold, NJ) at 37°C, the adventitia and intima were removed from the aortic medial layer, which was then placed in complete medium (Dulbecco's modified Eagle's medium containing 10% fetal bovine serum [FBS] and 50 μg/mL penicillin, 50 μg/mL streptomycin) for 2 hours. The arterial media was further digested with 2 mg/mL collagenase II/0.5 mg/mL elastase (Sigma) for 90 minutes at 37°C, and the isolated cells were washed and plated in complete medium. In all cases, >95% of cells stained positive for α‐smooth muscle actin (α‐SMA).
Early passages (p3‐p5) rat VSMCs were cultured both with and without PDGF‐BB (0, 10, and 25 ng/mL) for 24 hours and then were lysed for Western blot analysis.
Early passages (p3‐p5) rat VSMCs were cultured both with and without PDGF‐BB (0, 10, and 25 ng/mL) for 24 hours and then were lysed for Western blot analysis.
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