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Endo-growth media

Manufactured by Angio-Proteomie
Sourced in United States

Endo-growth media is a specialized cell culture medium designed to support the growth and maintenance of endothelial cells. It provides the essential nutrients and growth factors required for the proliferation and differentiation of endothelial cells in vitro.

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4 protocols using Endo-growth media

Human RPE cell lines, ARPE-19 and D407 (a generous gift from Dr. Richard Hunt, University of North Carolina, Chapel Hill), and cervical cancer cell line, HeLa, were cultured as previously reported [26] (link), [27] . Human Retinal Microvascular Endothelial Cells (hRMVECs) were purchased from Angio-Proteomie (Boston, MA) and were cultured in ENDO-growth media (Angio-Proteomie). Honokiol was dissolved in DMSO and was added to the cells. In control samples 0.1% of DMSO, corresponding to the DMSO concentration in the cells treated with highest honokiol concentration, was added. Cells were exposed to hypoxic condition (i.e. 1% O2, 5% CO2, and 94% N2) in a bactron anaerobic chamber as previously reported [26] (link), [27] . Since under physiological conditions posterior segment of the eye experiences an oxygen tension between 8–22 mm Hg (i.e. 1–4% O2) [28] (link), [29] (link), we performed all our cell-based hypoxia experiments at 1% oxygen. For the qPCR experiments, total RNA was isolated from RPE cells grown under normoxia, hypoxia, or hypoxia treated with honokiol and quantified as described in our previous publications [26] (link), [27] .
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HGECs were purchased from Anigio-Proteomie (Boston, MA) and subcultured in Endo-growth media (Angio-Proteomie, Boston, MA). Apoptosis was quantified using the in situ cell death detection kit by TUNEL assay (Chemicon-Millipore, Billerica, MA). After treatment with different concentrations of media with D-glucose (5 mM/L D-glucose; low-glucose, 30 mM/L D-glucose; high-glucose, and D-glucose [5 mM/L]+D-mannitol [25 mM/L]; osmotic control) including 10−6 M GNQWFI (which showed a nearly complete inhibition of VEGF/VEGFR1 interaction) [20] (link) for 72 hr, the number of TUNEL-positive cells was counted in 10 randomly chosen fields at a magnification of 400×. Western blot analysis was performed for PI3k, total-Akt, phospho-Ser473 Akt, total eNOS, phospho-Ser1177 eNOS, SOD1, SOD2 and β-actin with specific antibodies. siRNA targeting VEGFR1 and scrambled siRNA (siRNA control; Bioneer, Deajeon, Korea) were complexed with transfection reagent (Lipofectamin 2000; Invitrogen, Carlsbad, CA) in low-glucose media according to the manufacturer's instructions.
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Human glomerular endothelial cells (HGECs) were purchased from Anigio-Proteomie (Boston, MA, USA) and subcultured in endo-growth media (Angio-Proteomie). The HGECs were then exposed to low glucose or high glucose, with or without the additional 6-h application of resveratrol (50 μM). siRNAs, targeted to AdipoR1 and AdipoR2 and scrambled siRNA (siRNA cont) were complexed with transfection reagent (Lipofectamin 2000; Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. The proportion of apoptotic cells was determined using ApopTaq In Situ Apoptosis Detection kits, based on the TUNEL assay. To quantify staining proportions, approximately 20 views (×400 magnification) were randomly imaged from each slide (see the Additional file 1 for further details).
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HGECs were purchased from Anigio-Proteomie (Boston, MA, USA) and subcultured in endo-growth media (Angio-Proteomie). After treatment with medium containing different concentrations of d-glucose (5 mmol/L d-glucose (low glucose), 40 mmol/L d-glucose (high glucose), or 5 mmol/L d-glucose plus 35 mmol/L mannitol (osmotic control)) and 1, 10, or 50 µg/mL anthocyanin for 6 h, western blotting was performed for phospho-Thr172 AMPK, total AMPK, PPARα, PGC-1α, ERR-1α, PPARγ, phospho-ACC, total ACC, superoxide dismutase (SOD)-1, SOD-2, BCL-2, BAX, and β-actin with specific antibodies. To examine the effects of anthocyanins on other renal cells in high-glucose medium, we also used cultured NMS2 mesangial cells (see Additional file 1 for further details).
siRNAs targeted toward Ampkα1, Ampkα2, and Sirt1, and a non-specific scrambled siRNA (siRNA control) were complexed with a transfection reagent (Lipofectamine 2000; Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. The sequences of the siRNAs were: α1-AMPK, 5′-GCAUAUGCUGCAGGUAGAU-3′; α2-AMPK, 5′-CGUCAUUGAUGAUGAGGCU-3′; SIRT1, 5′-CACCUGAGUUGGAUGAUAU-3′; and scrambled siRNA, 5′-CCUACGCCACCAAUUUCGU-3′ (Bioneer, Daejeon, Korea). At 24 h after transfection, HGECs were exposed to 40 mmol/L glucose and 50 µg/mL anthocyanins for 24 h.
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