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.
Pdgf bb
Manufactured by BioLegend
Sourced in United States
PDGF-BB is a recombinant human platelet-derived growth factor homodimer. It is a signaling protein that plays a role in cell growth, cell division, and angiogenesis.
Automatically generated - may contain errors
Lab products found in correlation
C 22062, by PromoCell (1 mentions)
C 12533, by PromoCell (1 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (1 mentions)
Palbociclib, by Merck Group (1 mentions)
Easystrainer cell, by Greiner (1 mentions)
Dulbecco s modified eagle s medium, by Merck Group (1 mentions)
Liberase research grade, by Merck Group (1 mentions)
Tnf α, by BioLegend (1 mentions)
Tgf β, by BioLegend (1 mentions)
Etanercept, by Merck Group (1 mentions)
Etanercept, by Pfizer (1 mentions)
Epigenetics compound library, by Selleck Chemicals (1 mentions)
Dextran solution, by Merck Group (1 mentions)
Dnase 1, by Roche (1 mentions)
L glutamine, by BioLegend (1 mentions)
Collagenase dispase, by Roche (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Basic fibroblast growth factor (bfgf), by BioLegend (1 mentions)
4 protocols using pdgf bb
1
Isolation and Stimulation of Vascular Smooth Muscle Cells
2
Isolating and Culturing Synoviocytes for Cytokine and Therapeutic Response Analysis
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Synoviocytes were isolated from synovial tissue by using standard procedures (19 ). Synoviocytes from patients with RA were digested with Liberase TM Research Grade (Sigma–Aldrich) for 90 min at 37°C. The digested synoviocyte slurries were filtered through a 100 μm cell strainer (EASYstrainer™ Cell; Greiner Bio-One, Kremsmünster, Austria). Cells were suspended in the growth medium consisting of Dulbecco's modified Eagle's medium (Sigma–Aldrich) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin and incubated at 37°C in 5% CO2. Cells at passages 3 to 6 were used in all experiments.
For cytokine stimulation, cells were treated with or without various concentrations (1, 10, and 100 ng/mL) of PDGF-BB (BioLegend; San Diego, CA, USA), TGF-β (BioLegend), or TNF-α (BioLegend) for 2 days, as per our approved experimental design. The role of therapeutic drugs was determined by treating cells stimulated with cytokines with 10, 25, or 50 μg/mL etanercept (Pfizer, NY, USA) or 7.5 μM or 15 μM palbociclib (Sigma–Aldrich), or a combination of 25 μg/mL etanercept and 7.5 μM palbociclib for 1 day, as per our experimental design. These doses of cytokines and inhibitors were selected based on the findings of previous studies (20 (link)–23 (link)). WST-8 assays were performed to assess cell proliferation by using the Cell Counting Kit-8 (CK04; Dojindo).
For cytokine stimulation, cells were treated with or without various concentrations (1, 10, and 100 ng/mL) of PDGF-BB (BioLegend; San Diego, CA, USA), TGF-β (BioLegend), or TNF-α (BioLegend) for 2 days, as per our approved experimental design. The role of therapeutic drugs was determined by treating cells stimulated with cytokines with 10, 25, or 50 μg/mL etanercept (Pfizer, NY, USA) or 7.5 μM or 15 μM palbociclib (Sigma–Aldrich), or a combination of 25 μg/mL etanercept and 7.5 μM palbociclib for 1 day, as per our experimental design. These doses of cytokines and inhibitors were selected based on the findings of previous studies (20 (link)–23 (link)). WST-8 assays were performed to assess cell proliferation by using the Cell Counting Kit-8 (CK04; Dojindo).
3
Inhibition of HKMTs Modulates Orbital Fibroblast Proliferation
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GO orbital fibroblasts were plated at 5 × 103 cells/well onto 96-well plates with DMEM containing 1%FBS and antibiotics overnight. For the screening experiments, orbital fibroblasts were pre-incubated with selective HKMT inhibitors from Epigenetics Compound Library (Catalog No. L1900, Selleck Chemicals, Inc., TX) for 24 h and stimulated with 50 ng/ml of PDGF-BB (Biolegend Inc., USA), either with or without the HKMTs inhibitors for another 24 h. For further experiments on orbital fibroblast activation, only EZH2, G9a and DOT1L inhibitors were used as shown in Supplementary Table S2 . In addition, orbital fibroblasts were exposed to PDGF-BB and HKMTs inhibitors simultaneously for 24 h in an experimental set-up more representative for disease treatment as previously reported6 (link). Then, orbital fibroblast proliferation was determined based on uptake and subsequent release of methylene blue dye, as described previously26 (link). Percentages of proliferation were calculated by the following formula: (test sample/negative control) *100.
4
Isolation and Culture of Mouse Brain Microvascular Endothelial Cells and Oligodendrocyte Progenitor Cells
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As modified from a published method56 , brains were obtained from wild-type or Cav-1−/− mice at the age of 6–8 weeks with cerebellum and meninges removed. After homogenization, the homogenate of brains was centrifugated at 150 × g for 5 min. The pallet was then resuspended in a 15% dextran solution (mol wt 60,000–76,000, Sigma-Aldrich) and centrifugated at 400 × g to isolate blood vessel fragments. The pallet of fragments was digested in collagenase/dispase (1 mg/ml, Roche) and DNase I (10 μg/ml, Roche) at 37 °C. The dissociated BMECs were washed and seeded onto coated plates in DMEM/F12 media with 20% FBS, 1% PS, 1% endothelial cell growth supplement (SclenCell), 1% l-glutamine, 1% heparin, and 2 ng ml−1 bFGF (Biolegend). In vitro hypoxia was induced by either chronic CoCl2 (10 μM, Sigma-Aldrich) for 5 consecutive days or OGD treatment for 4 h. Primary mouse OPCs were isolated from pups of postnatal days 0–2 as reported57 (link). In brief, mixed glial cells were cultured in DMEM/F12 with 10% FBS and 1% PS for 7 days. Then the flasks were shaken on the orbital shaker at 200 × g for 1 h. After washed, the flasks continued to shake for another 18–20 h at 4 × g. OPCs were obtained and plated onto coated plates in DMEM/F12 media containing 1% BSA (Gibco), 20 ng ml−1 ITSS (Roche), 20 ng ml−1 PDGF-BB (Biolegend) and 20 ng ml−1 bFGF (Biolegend).
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