Human ESC-RPE authentication was performed as previously described [16 (link)]. Briefly, transepithelial electrical resistance (TEER) was triplicate measured with Millicell volt-ohm meter (Merck Millipore) [17 (link)]. The key RPE protein expression and localization were verified with indirect immunofluorescence labeling for zonula occludens-1 (ZO-1), claudin-3, claudin-19, sodium-potassium adenosine triphosphatase (Na+/K+-ATPase), bestrophin, and MER Proto-Oncogene, tyrosine Kinase (MERTK). Enzyme-linked immunoassay (ELISA) for pigment epithelium-derived factor (PEDF) was carried out from apical and basal media collected after overnight incubation and analyzed with the Human PEDF ELISA kit (BioVendor) following the manufacturer’s instructions. Phagocytosis assay was conducted with porcine photoreceptor outer segments (POS) by 4 h apical incubation at 37 °C in the presence of 10% fetal bovine serum (Thermo Fisher Scientific), followed by labeling with anti-rhodopsin antibody and tetramethylrhodamine (TRITC). The nuclei were counterstained with DAPI included in ProLong Gold mounting medium (Thermo Fisher Scientific). Images were acquired with an LSM 700-800 Confocal microscope (Carl Zeiss) and processed with the Zen 2.3 SP1 Black software (Carl Zeiss). All primary and secondary antibody details appear in Table S1 (Additional file 1 ).
Lsm 700 800 confocal microscope
Manufactured by Zeiss
The LSM 700-800 Confocal microscope is a state-of-the-art imaging system designed by Zeiss. It utilizes laser scanning technology to capture high-resolution, three-dimensional images of microscopic samples. The core function of this equipment is to provide detailed, optical sectioning of specimens, enabling researchers to visualize and analyze intricate cellular and subcellular structures.
Automatically generated - may contain errors
Lab products found in correlation
Millicell voltohmmeter, by Merck Group (2 mentions)
Human pedf elisa kit, by BioVendor (2 mentions)
Zen 2.3 sp1 black software, by Zeiss (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Prolong gold mounting medium, by Thermo Fisher Scientific (1 mentions)
A11029, by Thermo Fisher Scientific (1 mentions)
A 21245, by Thermo Fisher Scientific (1 mentions)
A21236, by Thermo Fisher Scientific (1 mentions)
A 11030, by Thermo Fisher Scientific (1 mentions)
A21445, by Thermo Fisher Scientific (1 mentions)
3 protocols using lsm 700 800 confocal microscope
1
Characterization of Human ESC-Derived RPE
2
Immunofluorescence Staining of Transfected HEK Cells
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The whole procedure was carried out at room temperature. Transfected HEK cells were fixed with PBS + 4% paraformaldehyde for 30 min, and blocked with PBS + 40 mM ammonium chloride (NH4Cl) (Applichem, #A3661) for 5 min. Cells were then permeabilized in PBS + 0.2% saponin (Sigma, #S7900) for 5 min, washed once (5 min) with PBS + 0.2% BSA (PBS-BSA), and incubated for 30 min with the antibody-containing supernatants. After 3 washes (5 min) with PBS-BSA, cells were incubated for 30 min in PBS-BSA with secondary anti-mouse or anti-human or anti-rabbit IgG conjugated to AlexaFluor 546, 647 or 488 (A-11030, A-21236, A-11029, A-21445, A-21245, 1:400, Life Technologies). After 3 washes (5 min) with PBS-BSA, cells were mounted in Möwiol (Fluka, #33480). Pictures were taken using a Zeiss LSM700/800 confocal microscope, with a 63x oil immersion objective.
3
Characterization of Human ESC-RPE Cells
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Human ESC-RPE authentication was performed as previously described [15] . Brie y, transepithelial electrical resistance (TEER) was triplicate measured with Millicell volt-ohm meter (Merck Millipore) [16] . Key RPE protein expression and localization was veri ed with indirect immuno uorescence labeling for zonula occludens-1 (ZO-1), claudin-3, claudin-19, sodium-potassium adenosine triphosphatase (Na + /K + -ATPase), bestrophin, and MER Proto-Oncogene, tyrosine Kinase (MERTK). Enzyme-linked immunoassay (ELISA) for pigment epithelium-derived factor (PEDF) was carried out from apical and basal media collected after overnight incubation and analyzed with the Human PEDF ELISA kit (BioVendor) following manufacturer's instructions. Phagocytosis assay was conducted with porcine photoreceptor outer segments (POS) by 4 hours apical incubation at 37°C in the presence of 10% fetal bovine serum (Thermo Fisher Scienti c), followed by labeling with anti-rhodopsin antibody and tetramethylrhodamine (TRITC). Nuclei were counterstained with DAPI included in ProLong Gold mounting medium (Thermo Fisher Scienti c). Images were acquired with an LSM 700-800 Confocal microscope (Carl Zeiss) and processed with the Zen 2.3 SP1 Black software (Carl Zeiss). All primary and secondary antibody details appear in Table S1 (Additional le 1).
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