human primary retinal pigment epithelial (hrpe) cells  (Lonza)

Journal: Frontiers in Cell and Developmental Biology

Article Title: Integration of human stem cell-derived in vitro systems and mouse preclinical models identifies complex pathophysiologic mechanisms in retinal dystrophy

doi: 10.3389/fcell.2023.1252547

Figure Lengend Snippet: Dram2 loss exacerbates toxicity-induced human RPE cell death in vitro (A) Experimental design of loss of DRAM2 experiments in human cells. hRPE, human primary retinal pigment epithelial (RPE) cells; KD, knockdown; A2E, N-retinylidene-N-retinylethanolamine; NaOI 3 , Sodium Iodate; hPSC, human pluripotent stem cells; KO, Knockout. (B) Representative image of hRPE after infection with lentivirus expressing DRAM2 shRNA and GFP (left) and knockdown efficiency of the two different DRAM2 shRNAs assessed by quantitative RT-PCR (qPCR) analysis of DRAM2 expression (right) (C) hRPE cell survival following treatment with A2E (30 μM; left) and NaIO 3 (5 mM; right). Dotted horizontal line marks the median survival (50% of the cells alive). (D) Differentiation of DRAM2 WT and KO human pluripotent stem cells (hPSCs) into RPE (hPSC-RPE) showing pigmentation (top row, BF: Bright Field) and expression of RPE maker ZO-1 (bottom row). Scale bars: 50 µm. (E) Representative immunofluorescent image (left) of RPE marker ZO-1 (green) in hPSC-RPE DRAM2 WT, KO1, and KO2 and phagocytosis of FITC-labeled Photoreceptor Outer Segments (POS; red). Scale bar: 20 µm. Phagocytosis capacity of the cells was assessed by quantification of cells with internalized FITC-POS by flow cytometry ( DRAM2 WT: blue line, DRAM2 KO1 and 2: orange lines). (F) hPSC-RPE cell survival following treatment with A2E (30 μM; left) and NaIO 3 (5 mM; right). Dotted horizontal line marks the median survival.

Article Snippet: Human primary retinal pigment epithelial (hRPE) cells (Lonza #00194987) were maintained in Retinal Pigment Epithelial Cell Growth Medium (RtEGM; Lonza) per manufacturer protocols.

Techniques: In Vitro, Knock-Out, Infection, Expressing, shRNA, Quantitative RT-PCR, Marker, Labeling, Flow Cytometry

Journal: Free radical biology & medicine

Article Title: 4-Hydroxy-7-oxo-5-heptenoic acid (HOHA) lactone induces apoptosis in retinal pigment epithelial cells

doi: 10.1016/j.freeradbiomed.2020.03.017

Figure Lengend Snippet: Generation of CEP epitopes in RPE cells under oxidative stress. (A) Images of the generation of CEP in RPE cells. (B) Quantification of the generation of CEP in ARPE-19 cells. (C) Quantification of the generation of CEP in hRPE cells. RPE cells were treated with 25 βM H2O2 for 2 hours followed by overnight recovery in a basal cell culture medium and then fixed in dry-ice cold acetone and immunostained with mouse monoclonal anti-CEP antibody (1°)/goat anti-mouse Alexa Fluor 488 antibodies (2°) and DAPI. Images were taken at 10X magnification with a Leica DMI 6000 B inverted fluorescence microscope. Image analysis was performed using Metamorph imaging software (Molecular Devices, Downington, PA).The figure is representative of three independent experiments that showed very similar results. ***P<0.0001

Article Snippet: CloneticsTM human primary retinal pigmented epithelial cells (hRPE) (passage 2) were obtained from Lonza (Allendale, NJ) and maintained and sub-cultured in RtEBM supplemented with an optimized mixture of growth factors and supplements (SingleQuotsTM Kit) and 10% heat-inactivated FBS. hRPE cells were passaged every 4–5 days.

Techniques: Cell Culture, Fluorescence, Microscopy, Imaging, Software

Journal: Free radical biology & medicine

Article Title: 4-Hydroxy-7-oxo-5-heptenoic acid (HOHA) lactone induces apoptosis in retinal pigment epithelial cells

doi: 10.1016/j.freeradbiomed.2020.03.017

Figure Lengend Snippet: Generation of CEP adducts in RPE cells under inflammatory stimulus. (A) Images of the generation of CEP in RPE cells. (B) Quantification of the generation of CEP in ARPE-19 cells. (C) Quantification of the generation of CEP in hRPE cells. RPE cells were treated with 12 μg/ml LPS for overnight and then immunostained with mouse monoclonal anti-CEP antibody/goat anti-mouse Alexa Fluor 488 antibodies and DAPI. Images were taken at 10X magnification with a Leica DMI 6000 B inverted fluorescence microscope. Image analysis was performed using Metamorph imaging software (Molecular Devices, Downington, PA). The figure shows a representative of three independent experiments, which showed very similar results. ***P<0.0001

Article Snippet: CloneticsTM human primary retinal pigmented epithelial cells (hRPE) (passage 2) were obtained from Lonza (Allendale, NJ) and maintained and sub-cultured in RtEBM supplemented with an optimized mixture of growth factors and supplements (SingleQuotsTM Kit) and 10% heat-inactivated FBS. hRPE cells were passaged every 4–5 days.

Techniques: Fluorescence, Microscopy, Imaging, Software

Journal: Free radical biology & medicine

Article Title: 4-Hydroxy-7-oxo-5-heptenoic acid (HOHA) lactone induces apoptosis in retinal pigment epithelial cells

doi: 10.1016/j.freeradbiomed.2020.03.017

Figure Lengend Snippet: Detection of ROS in RPE cells challenged with various concentrations of HOHA lactone. (A) Images of the generation of ROS in RPE cells. (B) Quantification of the generation of ROS in ARPE-19 cells. (C) Quantification of the generation of ROS in hRPE cells. Cells were treated with DCFHDA (15 μg/ml) in the basal medium under 5% CO2/95% air at 37 °C for 30 min, washed with a basal cell culture medium and then were exposed to HOHA lactone for two hours. Images were taken at 10X magnification with a Leica DMI 6000 B inverted fluorescence microscope. Image analysis was performed using Metamorph imaging software (Molecular Devices, Downington, PA). Data shown are representative of three independent experiments that showed very similar results. *P<0.05,***P<0.0001

Article Snippet: CloneticsTM human primary retinal pigmented epithelial cells (hRPE) (passage 2) were obtained from Lonza (Allendale, NJ) and maintained and sub-cultured in RtEBM supplemented with an optimized mixture of growth factors and supplements (SingleQuotsTM Kit) and 10% heat-inactivated FBS. hRPE cells were passaged every 4–5 days.

Techniques: Cell Culture, Fluorescence, Microscopy, Imaging, Software

Journal: Free radical biology & medicine

Article Title: 4-Hydroxy-7-oxo-5-heptenoic acid (HOHA) lactone induces apoptosis in retinal pigment epithelial cells

doi: 10.1016/j.freeradbiomed.2020.03.017

Figure Lengend Snippet: Cytotoxicity of HOHA lactone to (A) ARPE-19 cells and (B) primary hRPE cells (both cell types were seeded at 4.5×104 cells/well density) as measured by MTT assay. Eight replicate wells were used for each concentration of HOHA lactone in these studies. Results presented are percent cell survival in HOHA lactone treated groups with respect to vehicle (PBS)-treated cells (mean ± SD; n = 8). Data are representative of three independent experiments that show very similar results. *P<0.05, P<0.008, ***P<0.00001

Article Snippet: CloneticsTM human primary retinal pigmented epithelial cells (hRPE) (passage 2) were obtained from Lonza (Allendale, NJ) and maintained and sub-cultured in RtEBM supplemented with an optimized mixture of growth factors and supplements (SingleQuotsTM Kit) and 10% heat-inactivated FBS. hRPE cells were passaged every 4–5 days.

Techniques: MTT Assay, Concentration Assay