The human retinal epithelial cell line ARPE-19 was purchased from The Cell Bank of Type Culture Collection of Chinese Academy of Sciences and cultured in DMEM/F12 (HyClone; Cytiva) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) at 37˚C in an incubator with 95% air/5% CO2. In order to examine the effects of hyperglycemia, ARPE-19 cells were cultured with high concentration of glucose (33 mM; Sigma-Aldrich; Merck KGaA), normal concentration of glucose (5.5 mM) or 27.5 mM mannitol + 5.5 mM glucose as an osmotic control for 48 h. ARPE-19 cells were pretreated with nesfatin-1 (2.5 or 5 ng/ml; Sigma-Aldrich; Merck KGaA) for 1 h, followed by exposure to 33 mM glucose for 48 h (16 (link)).
Arpe 19
Manufactured by Merck Group
Sourced in United States
ARPE-19 is a human retinal pigment epithelial cell line. It is used for in vitro studies related to the retinal pigment epithelium.
Automatically generated - may contain errors
Lab products found in correlation
Arpe 19, by American Type Culture Collection (10 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Arpe 19, by Thermo Fisher Scientific (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
D glucose, by Merck Group (2 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (2 mentions)
Antibiotic antimycotic solution, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Biowest (1 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (1 mentions)
Pen strep, by PAN Biotech (1 mentions)
Fibronectin, by Innoprot (1 mentions)
Fungizone, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Innoprot (1 mentions)
Endothelial cell medium (ecm), by Innoprot (1 mentions)
Penicillin streptomycin, by Innoprot (1 mentions)
Ferric ammonium citrate, by Merck Group (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
18 protocols using arpe 19
1
Nesfatin-1 Mitigates Hyperglycemia-Induced ARPE-19 Cells
2
High Glucose Exposure in ARPE-19 Cells
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The human retinal pigmented epithelium cell line ARPE-19 was obtained from the American Type Culture Collection (ATCC, Manassas, VA). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) plus 10% fetal bovine serum (FBS, GIBCO, CA) at 37°C with 5% CO 2 . For HG treatment, the ARPE-19 cells were exposed to D-glucose (Sigma) ata concentration of 30 mmol/L in cultures as described in previous publications [28] . For normal glucose treatment, ARPE-19 cells were exposed to D-glucose (5.6 mmol/L) in cultures. For hypertonic treatment, ARPE-19 cells were exposed to D-glucose (5.6 mmol/L) and D-mannitol (Sigma, 24.4 mmol/L) in cultures.
3
ARPE-19 Cell Culture and Signaling Disruption
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A human RPE cell line (ARPE-19) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). ARPE-19 cells were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 medium containing 10% fetal bovine serum in a humidified atmosphere at 37°C with 5% CO2. The ARPE-19 cells were used at passages two to four in all the experiments and were treated with recombinant TGF-β2 (Santa Cruz Biotechnology, Santa Cruz, CAm USA) at a concentration of 10 ng/mL to simulate the PVR environment.
To disrupt the expression of EGFR, YAP, or RhoA, the ARPE-19 cells were treated with small interfering RNA (siRNA) against EGFR, YAP, or RhoA. EGFR-siRNA, YAP-siRNA, RhoA-siRNA and nonsilencing siRNA were obtained from Invitrogen (Carlsbad, CA, USA). Lipofectamine 2000 transfection reagent (ThermoFisher Scientific, Waltham, MA, USA) was used to perform transient transfection of siRNA. To disrupt EGFR signaling, ARPE-19 cells were treated with erlotinib (100 nM; Millipore) for 24 hours. To inhibit PI3K signaling, ARPE-19 cells were treated with LY294002 (25 µM; Bio-Rad Laboratories, Hercules, CA, USA) or wortmannin (100 nM; Bio-Rad Laboratories). To disrupt PDK1 signaling, ARPE-19 cells were treated with GSK2334470 (10 µM; Bio-Rad Laboratories). To disrupt Akt signaling, ARPE-19 cells were treated with MK-2206 (10 µM; Millipore).
To disrupt the expression of EGFR, YAP, or RhoA, the ARPE-19 cells were treated with small interfering RNA (siRNA) against EGFR, YAP, or RhoA. EGFR-siRNA, YAP-siRNA, RhoA-siRNA and nonsilencing siRNA were obtained from Invitrogen (Carlsbad, CA, USA). Lipofectamine 2000 transfection reagent (ThermoFisher Scientific, Waltham, MA, USA) was used to perform transient transfection of siRNA. To disrupt EGFR signaling, ARPE-19 cells were treated with erlotinib (100 nM; Millipore) for 24 hours. To inhibit PI3K signaling, ARPE-19 cells were treated with LY294002 (25 µM; Bio-Rad Laboratories, Hercules, CA, USA) or wortmannin (100 nM; Bio-Rad Laboratories). To disrupt PDK1 signaling, ARPE-19 cells were treated with GSK2334470 (10 µM; Bio-Rad Laboratories). To disrupt Akt signaling, ARPE-19 cells were treated with MK-2206 (10 µM; Millipore).
4
ARPE-19 Cell Glucose Exposure Protocol
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The human RPE cell line ARPE-19 was obtained from American Type Culture Collection (Manassas, VA, USA). The ARPE-19 cells were cultured in Dulbecco′s Modified Eagle′s Medium (Sigma-Aldrich, St. Louis, MO, USA) containing 10% fetal bovine serum (Biowest, Nuaillé, France) and 1% antibiotic antimycotic solution (ThermoFisher Scientific, Waltham, MA, USA) in a humidified 37 °C incubator with 5% CO2. The medium was changed every 2 days. We used cells between passages 16 and 24 for the experiments.
The ARPE-19 cells were maintained until 70–80% confluence and then exposed to serum-free starvation for 24 h, followed by treatment with NG (5.5 mM of D-glucose); OP control (5.5 mM of D-glucose plus 19.5 mM of mannitol); or HG (25 mM of D-glucose) for 48 h, unless stated otherwise. The experiments were conducted at least three times to confirm the consistency of the results.
The ARPE-19 cells were maintained until 70–80% confluence and then exposed to serum-free starvation for 24 h, followed by treatment with NG (5.5 mM of D-glucose); OP control (5.5 mM of D-glucose plus 19.5 mM of mannitol); or HG (25 mM of D-glucose) for 48 h, unless stated otherwise. The experiments were conducted at least three times to confirm the consistency of the results.
5
Cell Culture Protocols for Diverse Cell Lines
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Human embryonic kidney HEK-293T cells (ATCC-CRL-3216) and human immortalized keratinocyte HaCaT cells (94 (link)) were cultured in Dulbecco’s modified Eagle medium (DMEM; Gibco). Human retinal pigment epithelial cell line ARPE-19 (ATCC-CRL-2302) was cultured in Dulbecco’s modified Eagle medium/Nutrient Mixture F-12 Ham (D/F-12; SIGMA). All media were supplemented with 8% fetal bovine serum (FBS; SIGMA), penicillin-streptomycin (Pen/Strep; PAN-Biotech), and 2 mM Glutamine Stable (Cytogen). Cercopithecus aethiops kidney epithelial Vero cells (ATCC-CCL-81) were cultured in MEM Eagle (Cytogen) with 8% FBS. Cells were grown at 37°C, with 5% CO2 in a humidified incubator.
6
Culturing ARPE-19 and HREC Cells
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Human retinal pigment epithelial cells, ARPE-19 (CRL-2302, ATCC, Manassas, VA, USA), and human retinal endothelial cells, HREC (p10880, Innoprot, Vizcaya, Spain), were used. ARPE-19 cells (three passages) were grown to confluence (37 °C, 5% CO2) in Dulbecco’s modified Eagle’s medium (DMEM; D6429, Sigma-Aldrich, St. Louis, MO, USA) containing 10% fetal bovine serum (FBS; 10270106 Gibco ThermoFisher, Paisley, UK), 1% fungizone (Gibco, Carlsbad, CA, USA), and penicillin–streptomycin (Gibco, Carlsbad, CA, USA). HREC cells were seeded in T75 flasks (353136, Falcon, Corning Life Science, Tewksbury, MA, USA) covered with 1 mg/mL of fibronectin (Innoprot, p8248, Vizcaya, Spain) and grown to confluence in a standard incubator at 37 °C under humidified 5% CO2 conditions in Endothelial Cell Medium (Innoprot, p60104, Vizcaya, Spain) containing 5% FBS (Innoprot, Vizcaya, Spain), 1% Endothelial Cell Grow Supplement (ECGS, Innoprot, Vizcaya, Spain), and penicillin–streptomycin solution (Innoprot, Vizcaya, Spain).
7
Iron Modulation of ARPE-19 Cells
8
LINC00673 and p53 expression in h1RPE7 cells
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Human retinal RPEC cell line h1RPE7 and ARPE-19 were purchased from Sigma-Aldrich (USA) and cultured in DMEM: F12 media containing 10% FBS at 37 °C in a humidified incubator with 5% CO2.
LINC00673 and p53 expression pcDNA3 vectors were from GenePharma (Shanghai, China). Negative control (NC) siRNA and LINC00673 siRNA were from Sangon (Shanghai, China). h1RPE7 cells at 70–80% confluency (about 105 cells) were transfected with 10 nM LINC00673 and p53 expression pcDNA3 vector (empty pcDNA3 vector as NC group) or 45 nM LINC00673 siRNA (NC siRNA as NC group) using Lipofectamine 2000 reagent (Invitrogen, USA). Control cells were untransfected cells. Subsequent experiments were performed at 24h of post-transfection.
LINC00673 and p53 expression pcDNA3 vectors were from GenePharma (Shanghai, China). Negative control (NC) siRNA and LINC00673 siRNA were from Sangon (Shanghai, China). h1RPE7 cells at 70–80% confluency (about 105 cells) were transfected with 10 nM LINC00673 and p53 expression pcDNA3 vector (empty pcDNA3 vector as NC group) or 45 nM LINC00673 siRNA (NC siRNA as NC group) using Lipofectamine 2000 reagent (Invitrogen, USA). Control cells were untransfected cells. Subsequent experiments were performed at 24h of post-transfection.
9
ARPE-19 Cells: Caffeine and Inflammation
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Human retinal pigment epithelial cells (ARPE-19) were purchased from ATCC® (Manassas, Virginia, United States). Cells were cultured at 37°C (humidified atmosphere with 5% CO2) in ATCC-formulated DMEM:F12 medium (ATCC number 30–2006, Manassas, Virginia, United States) with 100 U/mL penicillin, 100 μg/ml streptomycin, and 10% fetal bovine serum (FBS). After reaching confluence (∼70%), ARPE-19 were pretreated for 24 h with caffeine at concentrations of 1, 10, 100 and 1000 µM (Sigma-Aldrich, Cat.No. C0750, St Louis, MO) and/or 1 µM of CGS 21680 hydrochloride (Tocris Bioscience, Cat.No. 1063, Bristol, United Kingdom) (Wang et al., 2014 (link)) in DMEM:F12 supplemented with only 5% FBS to starve cells. In control cells (untreated), only fresh medium has been added. After pretreatment, ARPE-19 were challenged with 150 ng/ml, 2 μg/ml, or 10 μg/ml of lipopolysaccharide E. coli (LPS) (Enzo Life Sciences ALX-581–010-L001, Farmingdale, NY) to simulate inflammation and also with different concentrations of caffeine (1–100 µM) and/or 1 µM of CGS 21680.
10
Cell Line Validation for PFCL Biocompatibility
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Two cell lines were included in the validation study: the ISO 10993-5 reference cell line, BALB/3T3, and ARPE-19 cell line, which simulate the cell type that comes in direct contact with PFCL during retina surgery. The murine fibroblast cells BALB/3T3 clone A31 (ATCC CCL163) and the human retinal pigment epithelial cell line ARPE-19 (ATCC CRL-2302) were obtained from the American Tissue Culture Collection (ATCC; Manassas, VA). After thawing, BALB/3T3 and ARPE-19 cells were grown in Dulbecco's modified eagle's medium (DMEM) with high glucose, L-glutamine, sodium pyruvate, and sodium bicarbonate (Sigma-Aldrich Corp., St. Louis, MO) and DMEM/Nutrient Mixture F-12 medium with L-glutamine without (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, Gibco, Monza, Italy), respectively, each supplemented with 10% of iron-fortified bovine calf serum (Sigma-Aldrich Corp.) and 1% penicillin-streptomycin (Sigma-Aldrich Corp.), as a monolayer in 75 cm2 culture flasks at 37°C ± 1°C, 90% ± 10% humidity, and 5.0% ± 1.0% CO2/air. The medium was changed every 48 hours. At 80% confluence, cells were removed from the flask by trypsinization. Single cell suspension was counted manually using trypan blue staining and a hemocytometer. The counting was repeated before each cell passage or seeding in 96-well plates. After thawing, cells were passaged two times before using them in the test.
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