Human umbilical vein endothelial cells (HUVEC) were isolated by collagenase 0.25 mg/mL, Collagenase Type II from Clostridium histolyticum (Boehringer, Mannheim, Germany) digestion from umbilical cords obtained at birth from pregnancies and cultured (37°C, 5% CO2) in 1% gelatin-coated petri dishes up to passage 2 in medium 199 (M199; Life Technologies, Carlsbad, CA, USA) containing 5 mmol/L D-glucose, 10% new born bovine serum, 10% fetal bovine serum (FBS), 3.2 mmol/L L-glutamine, and 100 U/mL penicillin-streptomycin (primary culture medium). A7r5 cells, a vascular smooth muscle cell (VSMC) line originally derived from embryonic rat aorta, were purchased from the American Type Culture Collection (ATCC). They were cultured in Dulbecco's Modified Eagle Medium (DMEM; Invitrogen, NY, USA, Sigma-Aldrich) supplemented with 10% FBS and 2 × 10−3 M pyruvate. Prior to experiments, 80–90% confluent A7r5 cells were serum-starved.
Collagenase type 2 from clostridium histolyticum
Manufactured by Boehringer Ingelheim
Sourced in Germany
Collagenase Type II from Clostridium histolyticum is an enzyme used in research and laboratory settings. It is derived from the bacterium Clostridium histolyticum and its primary function is the digestion of collagen, a structural protein found in various tissues.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (1 mentions)
A7r5 cells, by American Type Culture Collection (1 mentions)
Ng nitro l arginine methyl ester l name, by Merck Group (1 mentions)
Immobilon p polyvinylidene difluoride membranes, by Bio-Rad (1 mentions)
4 amino 5 methylamino 2 7 difluorofluorescein daf fm, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Anti β actin, by Merck Group (1 mentions)
2 protocols using collagenase type 2 from clostridium histolyticum
1
Isolation and Culture of HUVEC and A7r5 Cells
2
Immunoblotting of Phosphorylated eNOS
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Primary monoclonal mouse anti-eNOS phosphorylated at serine 1177 , anti-eNOS phosphorylated at threonine 495 , and anti-β-actin were from Sigma Aldrich (St Louis, MO, USA). Primary monoclonal mouse antitotal eNOS antibody and secondary horseradish peroxidase-conjugated goat anti-mouse antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). For isolation of HUVECs from umbilical cords, Collagenase Type II from Clostridium histolyticum (Boehringer, Mannheim, FRG) was used. Medium M199, newborn (NBCS) and foetal calf (FCS) sera, L-glutamine, and penicillin-streptomycin were from Gibco Life Technologies (Carlsbad, CA, USA). L-[ 3 H]Arginine and D-[ 3 H]mannitol were from NEN (Dreieich, FRG). N G -Nitro-L-arginine methyl ester (L-NAME) was from Sigma Aldrich, Immobilon-P polyvinylidene difluoride membranes from BioRad Laboratories (Hertfordshire, UK), and the fluorescent dye 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM) from Molecular Probes (Leiden, The Netherlands).
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