Reduced L-glutathione, sodium borohydride, 2-methyl-2-butene solution (2.0 M in THF), sodium chlorite, tert-butanol, glutathione reductase from baker’s yeast (250 units/ml. Sigma catalogue #G3664), 5,5’-dithio-bis(2-nitrobenzoic acid) (DTNB), and NADPH were purchased from Sigma-Aldrich (St. Louis, MO). HOHA-lactone (1 ) was prepared as described previously.1 (link) N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) was obtained from Acros Organics (Thermo Fisher, New Jersey). Dulbecco’s modified Eagle’s medium, high glucose (DMEM/F12), fetal bovine serum, penicillin–streptomycin and L-glutamine were obtained from Gibco (Life Technologies, Grand Island, NY). ARPE-19 cells were obtained from American Type Culture Collection (ATCC, Rockville, MD). The hRPE cells were obtained from Lonza Walkersville, Inc., Walkersville, MD, catalog # 00194987, lot # 0000424472. According to the manufacturer, they exhibited pan-cytokeratin staining (95%), anti-fibroblast staining (0%) and ZO-1 staining (strongly positive), and they have viability >70%, average doubling time 22 hrs. Retinal pigmented epithelial cell basal medium, an optimized mixture of growth factors and supplements for primary hRPE cells (SingleQuots™ Kit), was obtained from Lonza (Allendale, NJ).
Hrpe cells
Manufactured by Lonza
Sourced in United States
HRPE cells are a type of primary human retinal pigment epithelial cells derived from human donor eyes. They are used in cell culture research and serve as a model system for studying the physiology and function of the retinal pigment epithelium, a crucial layer of cells in the eye.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
5 5 dithiobis 2 nitrobenzoic acid dtnb, by Merck Group (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Arpe 19 cell, by American Type Culture Collection (1 mentions)
Singlequots kit, by Lonza (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Nadph, by Merck Group (1 mentions)
High glucose dmem f12, by Thermo Fisher Scientific (1 mentions)
Rtegm singlequots, by Lonza (1 mentions)
Primary antibody against β actin, by Santa Cruz Biotechnology (1 mentions)
Secondary antibodies of the horseradish peroxidase hrp conjugated goat anti rabbit igg, by Santa Cruz Biotechnology (1 mentions)
Enhanced chemiluminescent substrate for detection of hrp, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Arpe 19, by Thermo Fisher Scientific (1 mentions)
Rtegm bulletkit, by Lonza (1 mentions)
Lps escherichia coli o111 b4, by Merck Group (1 mentions)
Ham s f 12 nutrient mixture, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium, by Merck Group (1 mentions)
5 protocols using hrpe cells
1
Glutathione-Mediated Oxidative Stress Assay
2
Culturing Human Retinal Pigment Epithelial Cells
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Human RPE (hRPE) cells (Lonza, Walkersville, MD, USA) were cultured in RPE cell basal medium with RtEGM SingleQuots (Lonza) containing 2% fetal bovine serum (FBS), l -glutamine, fibroblast growth factor (FGF), and GA-1000 in a 37 °C humidified atmosphere with 5% CO2 according to the manufacturer's recommendations. All experiments were performed using fourth passage cells.
3
Modulation of p53 and MDM2 Regulation
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The antibodies against p53 and MDM2 were purchased from Cell Signaling Technology (Danvers, MA, USA) and from Abgent (San Diego, CA, USA), respectively. The primary antibody against β-actin and the secondary antibodies of the horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG and anti-mouse IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Enhanced chemiluminescent substrate for detection of HRP was from Thermo Scientific (Waltham, MA, USA).
hRPE cells were purchased from Lonza (Walkersville, MD, USA) and cultured in Dulbecco's modified Eagle's medium (DMEM)/nutrient mixture F-12 medium (F12) (Thermo Scientific, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. The hRPE cells with MDM2 T309G or T309T only were created using a system of AAV-SpCas9 D10A and AAV-SpGuide (MDM2 or lacZ) and a homology direct recombinant DNA template with MDM2 G309 as described previously.21 (link) All cells were cultured at 37°C in a humidified 5% CO2 atmosphere.
hRPE cells were purchased from Lonza (Walkersville, MD, USA) and cultured in Dulbecco's modified Eagle's medium (DMEM)/nutrient mixture F-12 medium (F12) (Thermo Scientific, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. The hRPE cells with MDM2 T309G or T309T only were created using a system of AAV-SpCas9 D10A and AAV-SpGuide (MDM2 or lacZ) and a homology direct recombinant DNA template with MDM2 G309 as described previously.21 (link) All cells were cultured at 37°C in a humidified 5% CO2 atmosphere.
4
ARPE-19 and Primary hRPE Cell Culture
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This study used the human RPE cell line ARPE-19 (CLR-2302; American Type Culture Collection, Manassas, VA, USA) between 25 and 27 passages and primary human RPE (hRPE) cells (Lonza, Walkersville, MD, USA) between 5 and 6 passages. ARPE-19 cells were cultured in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin/100 µg/mL streptomycin (P/S) and passaged at ratios of 1:2 to 1:4 using trypsin–EDTA (Thermo Fisher Scientific, Waltham, MA, USA). Primary hRPE cells were cultured in basal media containing supplements (RtEGM BulletKit; Lonza). For subculture, 2% FBS was added to the media, and the media were replaced with serum-free media after 24 hours. Cells were treated with 10 µg/mL LPS (Escherichia coli O111:B4; Sigma-Aldrich, St. Louis, MO, USA) with or without fursultiamine–HCl (Toronto Research Chemicals, North York, ON, Canada) at the specified concentrations. Confluent ARPE-19 cells were maintained in a medium consisting of DMEM/F12, 1% FBS, 1.5 mM pyruvate, and 1% P/S for mitochondrial activation, as described previously.18 (link) The medium was changed every 2 days through days 4 to 6.
5
Primary Human RPE Cell Culture and Transfection
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Primary human RPE (hRPE) cells were purchased from Lonza (Walkersville, MD, USA) and used for in vitro assays. The cells were grown in the Dulbecco's modified Eagle's medium premixed with Ham's F-12 nutrient mixture (1:1 ratio; Sigma-Aldrich Corp., St. Louis, MO, USA) and supplemented with 10% fetal bovine serum (FBS) and streptomycin/penicillin G antibiotics (Sigma-Aldrich Corp.). The primary hRPE cells were transfected with Stealth small interfering RNA (siRNA; Invitrogen, Carlsbad, CA, USA) targeting CAVEOLIN-1 (HSS141466) and the negative control (siRNA_Ctrl). The primary mouse RPE (mRPE) cells were collected from the wild-type mice and the Cav-1 À/À mice as previously described. 20, 21
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