RPE cells from VMD2-Cre;Vhlfl/fl and control Vhlfl/fl littermates were isolated as previously described (Krohne et al., 2012 (link)). Cells were maintained at 37° and 5% CO2 in DMEM/F12 from Thermo Fisher Scientific (Waltham, MA) with 2% FBS from Jackson ImmunoResearch (West Grove, PA). 2 μg/μl doxycycline was added to all cultures daily for three days to induce Vhl ablation. Human RPE cells (Lonza) were maintained either on plastic surfaces or on transwell filters (Corning) depending on the application in RtEGM Retinal Pigment Epithelial Cell Growth Medium fortified with RtEGM BulletKit from Lonza (Basel, Switzerland). Glucose, lactate, and glutamine levels were measured from the media in apical and basal compartments using a 2900 Biochemistry Analyzer from YSI (Yellow Springs, OH). A concentrated stock of DMOG from Cayman Chemical (Ann Arbor, MI) was made with DMSO, and added directly to the media in different concentrations to induce pseudo-hypoxia. RPE cells were maintained in low oxygen and metabolic changes were analyzed using Seahorse Flux Analysis (see below) in a Coy Dual Hypoxia Chamber from Coy Lab Products (Grass Lake, MI) (Grassian et al., 2014 (link)).
Rtegm retinal pigment epithelial cell growth medium
Manufactured by Lonza
Sourced in Switzerland
RtEGM Retinal Pigment Epithelial Cell Growth Medium is a specialized cell culture medium designed for the growth and maintenance of retinal pigment epithelial (RPE) cells. The medium provides the necessary nutrients and growth factors to support the in vitro culture of RPE cells.
Automatically generated - may contain errors
Lab products found in correlation
Dmem f12, by Thermo Fisher Scientific (3 mentions)
Rtegm bulletkit, by Lonza (1 mentions)
Human rpe cells, by Lonza (1 mentions)
Fetal bovine serum (fbs), by Jackson ImmunoResearch (1 mentions)
Transwell filter, by Corning (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Egm 2, by Lonza (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
3 protocols using rtegm retinal pigment epithelial cell growth medium
1
Isolation and Metabolic Analysis of RPE Cells
2
Isolation and Knockdown of Choroidal Endothelial Cells
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Choroidal endothelial cells were isolated from young donor eyes obtained from the Utah Zions Eye Bank (Salt Lake City, UT, USA), as previously reported.18 (link) Endothelial cell (EC) phenotype was confirmed by labeling of EC markers (CD31, von Willebrand factor [vwF], and vascular endothelial [VE]-cadherin), and CECs of passages 2 through 5 were used in experiments. Choroidal endothelial cells were maintained in endothelial growth medium-2 (EGM-2; Lonza, Walkersville, MD, USA) with 5% fetal bovine serum (FBS). Human RPE cells were obtained from Lonza and grown in medium (RtEGM Retinal Pigment Epithelial Cell Growth Medium; Lonza) plus 2% FBS and penicillin-streptomycin. Cells below passage 5 were used for experiments.
To knockdown Thy-1, CECs were transfected using reagent (Lipofectamine 2000; Life Technologies, Grand Island, NY, USA) per the commercial protocol, with siRNA targeting the human Thy-1 gene or silencer selective negative control siRNA (both from Life Technologies). Forty-eight hours after transfection, cells were analyzed.
To knockdown Thy-1, CECs were transfected using reagent (Lipofectamine 2000; Life Technologies, Grand Island, NY, USA) per the commercial protocol, with siRNA targeting the human Thy-1 gene or silencer selective negative control siRNA (both from Life Technologies). Forty-eight hours after transfection, cells were analyzed.
3
Evaluation of Nutlin-3, NUT, and LIPO-NUT on Retinal Cells
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The biological effects of Nutlin-3, NUT and LIPO-NUT were firstly evaluated on ARPE-19 cell line, a human retinal pigment epithelial cell line. This cell line was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and was cultured in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12) added with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin and 100 mg/mL streptomycin (all from Gibco, Grand Island, NY, USA). Cells were maintained at 37 °C with a 5% CO2 and 90% relative humidity atmosphere.
Subsequently, the effects of selected LIPO-NUT were also tested on human primary retinal pigmented epithelium (HRPE) cells. HRPE cells were obtained from Lonza (Basel, Switzerland) and were maintained in RtEGM™, Retinal Pigment Epithelial Cell Growth Medium (Lonza), containing its specific supplements (L-glutamine, GA-1000, human FGF-B and 2%FBS) at 37 °C with a 5% CO2 and 90% relative humidity atmosphere. HRPE cells were cultured and used for experiments within the fifth passage.
Subsequently, the effects of selected LIPO-NUT were also tested on human primary retinal pigmented epithelium (HRPE) cells. HRPE cells were obtained from Lonza (Basel, Switzerland) and were maintained in RtEGM™, Retinal Pigment Epithelial Cell Growth Medium (Lonza), containing its specific supplements (L-glutamine, GA-1000, human FGF-B and 2%FBS) at 37 °C with a 5% CO2 and 90% relative humidity atmosphere. HRPE cells were cultured and used for experiments within the fifth passage.
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