Human VSMCs (ATCC, Manassas, VA, USA) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS), glutamine and penicillin/streptomycin at 37 °C in an humidified atmosphere of 5% CO2. VSMCs were transfected with miR-455 inhibitor or si-circ-ATL1 at a concentration of 50 nM using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. For SIRT5 overexpression, VSMCs were transfected with 1 μg SIRT5 plasmid or control vector. Transfected cells were used in subsequent experiments 2 days after transfection.
Human vsmcs
Manufactured by American Type Culture Collection
Sourced in United States
Human vascular smooth muscle cells (VSMCs) are primary cells derived from human vascular tissue. They are a critical component of the vascular system and play a key role in maintaining vascular tone and structure.
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Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Huvecs, by PromoCell (1 mentions)
Ham s f 12k medium, by Thermo Fisher Scientific (1 mentions)
Mir 183 5p mimic, by RiboBio (1 mentions)
Agomir nc, by Thermo Fisher Scientific (1 mentions)
Antagomir nc, by Thermo Fisher Scientific (1 mentions)
Agomir nc, by RiboBio (1 mentions)
Antagomir nc, by RiboBio (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Smooth muscle cell growth supplement, by Thermo Fisher Scientific (1 mentions)
Dharmafect 1 reagent, by GE Healthcare (1 mentions)
Mir 25 mimic, by GenePharma (1 mentions)
Quikchange site directed mutagenesis kit, by Agilent Technologies (1 mentions)
7 protocols using human vsmcs
1
VSMC Transfection and SIRT5 Overexpression
2
Culturing HUVECs and Human VSMCs
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HUVECs (Promocell, Heidelberg, Germany) were cultured in endothelial basal medium supplemented with hydrocortisone, bovine brain extract, epidermal growth factor, gentamycin and 10% fetal bovine serum at 37 °C in a humidified atmosphere of 95% air and 5% CO2. Human VSMCs (ATCC, Rockville, MD, USA) were cultured in Kaighn's modified Hams F12 medium supplemented with 10 FBS and 50% VSMC growth medium.
3
Ox-LDL-Induced In Vitro Atherosclerosis Model
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Human VSMCs (ATCC, USA) were grown in Ham's F-12 K medium (Gibco/BRL life Technologies, Germany) containing 10 % fetal bovine serum (Hyclone, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin, and placed under humidified conditions of 37 °C, 5% CO2. VSMCs were treated with 50 μg/mL Ox-LDL (Peking Union Biology Company, China) for 6 h, 12 h, and 24 h to establish an in vitro AS model.14 (link)
4
Modulating miR-183-5p Expression in VSMCs
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Human VSMCs were purchased from the American Type Culture Collection. Cells were grown in the Dulbecco's modified Eagle's medium (DEME; Hyclone, GE Healthcare, Logan, UT, USA) with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Waltham, MA, USA). Then cells were incubated in a humidified atmosphere with 5% CO 2 at 37°C. MiR-183-5p mimic, miR-183-5p inhibitor, and their relative control miRNAs (mimic NC and inhibitor NC) were provided by Ribobio (Guangzhou, China). The transfections were performed to regulate the expression level of miR-183-5p by using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Forty-eight hours post transfection, cells were collected for further experiments.
5
MiR-183-5p Regulation in VSMCs
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Human VSMCs were purchased from the American Type Culture Collection (ATCC). Cells were grown in the Dulbecco's modi ed Eagle's medium (DMEM; Hyclone, GE Health Care, USA) with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scienti c, Waltham, USA). Then cells were incubated in a humidi ed atmosphere with 5% CO 2 at 37℃. MiR-183-5p agomir, miR-183-5p antagomir, and their relative control miRNAs (agomir NC and antagomir NC) were provided by Ribobio (Guangzhou, China). The sequences were as follows: miR-183-5p agomirs, 5'-UAUGGCACUGGUAGAAUUCACU-3'; miR-183-5p antagomir, 5'-AGUGAAUUCUACCAGUGCCAUA-3'; agomirs NC, 5'-UGUAGGGCCACUCAGUCAACUU-3'; antagomir NC, 5'-AAUAUGGGCGAAAUGGGGCCAUC-3'.
As previous study described, cells were transfected with 100 nM miR-183-5p agomir or antagomir, or agomir NC, antagomir NC to regulate the expression level of miR-183-5p by using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions [18] . 48h post-transfection, cells were collected for further experiments. To investigate ox-LDL regulation on miR-183-5p expression, VSMCs were treated with ox-LDL (40 μg/ml).
As previous study described, cells were transfected with 100 nM miR-183-5p agomir or antagomir, or agomir NC, antagomir NC to regulate the expression level of miR-183-5p by using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions [18] . 48h post-transfection, cells were collected for further experiments. To investigate ox-LDL regulation on miR-183-5p expression, VSMCs were treated with ox-LDL (40 μg/ml).
6
Overexpression of LIPCAR in VSMCs
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Human VSMCs obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) were human aorta smooth muscle. These VSMCs were originated from an 11-month-old white female. The culture medium was Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) containing 10% fetal bovine serum (FBS, Gibco, USA), incubated at 37°CC in a humidified atmosphere with 5% CO2. Cells were treated with ox-LDL (20 μg/ml) or PDGF-BB (20 ng/ml) for 24 h. The overexpression plasmid pcDNA-LIPCAR and negative control plasmid pcDNA-control were designed and purchased from Hanbio (Shanghai, China). Lipofectamine® 2000 (Thermo Fisher Scientific, Inc.) was used to transfect plasmids into VSMCs. Cells transfected for 48 h were used for further research.
7
miR-25 Regulation of CDK6 in VSMCs
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All experiments were approved by the Clinical Research Ethics Committee of The Fourth Affiliated Hospital, Harbin Medical University (Harbin, China).
Vectors and cell culture. The pcDNA-CDK6 vector was purchased from Sigma-Aldrich (Oakville, ON, Canada). The CDK6 3'UTR sequence with the binding site for miR-25 was cloned into the pMIR-REPORT luciferase construct (Ambion Life Technologies, Carlsbad, CA, USA). The mutant construct of CDK6 3'UTR was generated using the QuikChange Site-Directed Mutagenesis kit (Agilent Technologies, Inc., Santa Clara, CA, USA). The reagents for cell culture (fetal bovine serum, penicillin and streptomycin) were purchased from Gibco Life Technologies (Carlsbad, CA, USA). Human VSMCs were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in the medium 231 supplemented with smooth muscle cell growth supplement (Gibco Life Technologies) at 37˚C in a humidified atmosphere of 95% air and 5% CO 2 .
Cell transfection. The miR-25 mimics and the control were synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China) and transfected into the cells to a final oligonucleotide concentration of 20 nmol/l. All cell transfections were introduced using DharmaFECT 1 reagent (GE Healthcare Biosciences, Pittsburgh, PA, USA) according to the manufacturer's instructions.
Vectors and cell culture. The pcDNA-CDK6 vector was purchased from Sigma-Aldrich (Oakville, ON, Canada). The CDK6 3'UTR sequence with the binding site for miR-25 was cloned into the pMIR-REPORT luciferase construct (Ambion Life Technologies, Carlsbad, CA, USA). The mutant construct of CDK6 3'UTR was generated using the QuikChange Site-Directed Mutagenesis kit (Agilent Technologies, Inc., Santa Clara, CA, USA). The reagents for cell culture (fetal bovine serum, penicillin and streptomycin) were purchased from Gibco Life Technologies (Carlsbad, CA, USA). Human VSMCs were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in the medium 231 supplemented with smooth muscle cell growth supplement (Gibco Life Technologies) at 37˚C in a humidified atmosphere of 95% air and 5% CO 2 .
Cell transfection. The miR-25 mimics and the control were synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China) and transfected into the cells to a final oligonucleotide concentration of 20 nmol/l. All cell transfections were introduced using DharmaFECT 1 reagent (GE Healthcare Biosciences, Pittsburgh, PA, USA) according to the manufacturer's instructions.
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