Human aortic SMCs and adventitial fibroblasts were purchased from Promo Cell (Catalog No: C-12533, C-12380) and cultured in growth medium (Promo Cell, Catalog No: C39262). The SMC and fibroblasts cell purity were verified using a SMC α-actin (1:100, Abcam catalog No: ab5694) and ERTR7 (1:100, Abcam catalog No: ab5694) antibodies by immunohistochemistry. Cells of passages 2–3 were used for the short interfering (si)RNA transfection study. SMCs were transfected with control siRNA or siRNA targeting human calpain-2 (Ambion-Silencer Select validated siRNA, ThermoFisher Scientific) sequences using RNAiMax lipofectamine transfection reagent (Life Technologies, ThermoFisher Scientific). After 48 h of transfection, cells were incubated with either vehicle or AngII (1 μM) for 24 h; then protein lysates were harvested for western blot analyses.
C 12533
Manufactured by PromoCell
The C-12533 is a laboratory equipment product. It is designed for general laboratory use. The core function of this product is to perform tasks typically required in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Ambion silencer select validated sirna, by Thermo Fisher Scientific (1 mentions)
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (1 mentions)
C 22062, by PromoCell (1 mentions)
Pdgf bb, by BioLegend (1 mentions)
Am16708, by Thermo Fisher Scientific (1 mentions)
Am4613, by Thermo Fisher Scientific (1 mentions)
Recombinant tgf β1, by R&D Systems (1 mentions)
Sb431542, by R&D Systems (1 mentions)
4 protocols using c 12533
1
Aortic SMC and Adventitial Fibroblast Calpain-2 Regulation
2
Isolation and Stimulation of Vascular Smooth Muscle Cells
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Primary murine VSMCs were isolated from mouse aorta and routinely maintained in DMEM supplemented with 10% FBS, as described in our previous study.8 , 24 Human aortic SMCs were purchased from PromoCell GmbH (C‐12533) and cultured in SMC growth medium 2 (C‐22062; PromoCell GmbH), according to the manufacturer's instructions. VSMCs between passages 5 and 10 were used in the current study. VSMCs were treated with various atherogenic stimuli, as described in the previous studies.9 , 25 , 26 , 27 , 28 Briefly, for platelet‐derived growth factor BB (PDGF‐BB; BioLegend) and serum stimulation, VSMCs were serum starved for 24 to 48 hours (0.5% FBS), followed by an incubation with 20% FBS and 10 ng/mL PDGF‐BB for 3, 6, 12, 24, and 48 hours. For oxidized low‐density lipoprotein component treatments, VSMCs were serum starved for 24 to 48 hours, followed by incubation with 10 μmol/L 4‐hydroxynonenal and 7‐ketocholesterol for 24 hours.
3
MFAP4 Knockdown in Human Aortic SMCs
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Human aortic SMCs (PromoCell, C-12533, 3 different donors) were cultured on 6-well plates (1.5×105 cells/well) using SMC Growth Medium 2 (PromoCell C-22062) until 70% confluent. MFAP4 siRNA (50 nmol/L, Life Technologies, AM16708, Assay ID: 11386) and control siRNA (50 nmol/L, Life Technologies, AM4613) were transfected by using lipofectamine in different passages of different donors (2 for donor 1, 7 for donor 2, and 5 for donor 3) for 6 hours and subsequently in M199 with 100 U/mL penicillin, 100 µg/mL streptomycin, and 2.5% fetal bovine serum overnight. After replaced with complete medium (M199 with 100 U/mL penicillin, 100 µg/mL streptomycin, and 20% fetal bovine serum) and cultured for 48 hours, the cells were washed using M199 3 times, and further cultured in M199 for 24 hours. The cells were harvested for RNA extraction as detailed below. In total, 14 individual experiments using 3 different cell lines were performed for each condition. The experiment was repeated with a different MFAP4 siRNA at a lower concentration (25 nmol/L, Life Technologies, 4392420, Assay ID: s8716).
4
Primary Aortic Smooth Muscle Cell Culture and TGF-β1 Modulation
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Primary human aortic SMCs (PromoCell, C-12533, 3 different donors, see Major Resources Table in the online-only Data Supplement ) were cultured in DMEM with 2 mmol/L L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 20% fetal bovine serum. Murine aortic SMCs were cultured similarly, yet with DMEM/F12, from wild-type C57Bl6/J aortas after enzymatic digestion as described previously.25 (link) Cell experiments were initiated with a subconfluent SMC layer at passage 6 to 10. SMCs were preincubated with serum-free medium for 24 hours, followed by a 24-hour incubation with 10 ng/mL recombinant TGF-β1 (R&D, 240-B-002) with or without 10 µmol/L of the ALKi (activin receptor-like kinase 4,5,7 inhibitor) SB 431542 (R&D, 1614) to block TGF-β type I receptors. Independent culture experiments were performed and normalized to the controls (no TGF-β1 and no ALKi) for comparison of fold change after TGF-β1 or simultaneous TGF-β1 and ALKi treatment.
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