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 ).
Zen 2.3 sp1 black software
Manufactured by Zeiss
Sourced in Germany
Zen 2.3 SP1 Black is a software package developed by Zeiss for use with their microscopy and imaging equipment. The software provides a platform for controlling and managing various microscopy functions and imaging workflows. It offers a range of tools and features to facilitate image acquisition, processing, and analysis. The core function of the software is to enable users to efficiently operate and integrate Zeiss microscopy systems into their research or workflow.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Millicell voltohmmeter, by Merck Group (2 mentions)
Human pedf elisa kit, by BioVendor (2 mentions)
Lsm 700 800 confocal microscope, by Zeiss (2 mentions)
Plan apo 100 oil immersion lens, by Zeiss (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Prolong gold mounting medium, by Thermo Fisher Scientific (1 mentions)
Epidermal growth factor (egf), by Miltenyi Biotec (1 mentions)
Fibroblast growth factor 2 (fgf2), by Miltenyi Biotec (1 mentions)
Human fgf 2, by Miltenyi Biotec (1 mentions)
Ultra low attachment 6 well plate, by Corning (1 mentions)
Penicillin streptomycin, by Biowest (1 mentions)
Human egf, by Miltenyi Biotec (1 mentions)
6 protocols using zen 2.3 sp1 black software
1
Characterization of Human ESC-Derived RPE
2
FRAP Assay for Biomolecular Condensates
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FRAP was performed using Zen software on a Zeiss LSM710 confocal microscope equipped with a 1.46 numerical aperture (NA) Plan-Apo ×100 oil-immersion lens (Carl Zeiss, Germany). The droplets were scanned five times before photobleaching. Most photobleaching experiments were performed by exposing the region of interest 100 times at 75% intensity of a 488 nm wavelength of an argon laser. The photobleached region was then allowed to recover for 75 cycles, with laser power attenuated to 1.5% intensity. FRAP assays of droplets formed with EPYC1/EPYC1-GFP using SeL8 (Supplementary Fig. 6e ) was performed using 100% intensity of the 488 nm laser and allowed to recover for 35 cycles. The area bleached was a 1.25 µm2 circle (1.26 µm diameter) or varied with droplet size in case the whole droplet was bleached.
Image acquisition and analysis were performed on Zen 2.3 SP1 (black) software from Carl Zeiss. Data were excluded from analysis when droplets fused (causing a spike in fluorescence intensity).
Image acquisition and analysis were performed on Zen 2.3 SP1 (black) software from Carl Zeiss. Data were excluded from analysis when droplets fused (causing a spike in fluorescence intensity).
3
CSC Sphere Formation and Quantification
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In total, 100,000 cells were seeded in a non-adherent plates with CSC medium (DMEM F12 supplemented with 1X B27™ (ThermoFisher Scientific, Waltham, MA, USA), 10 ng/mL Fibroblast Growth Factor-2 (Miltenyi Biotec, Bergisch Gladbach, Germany), and 10 ng/mL Epidermal Growth Factor (Miltenyi Biotec, Bergisch Gladbach, Germany). Medium was renewed every 4 days by low speed centrifugation (5 min, 800 rpm). Tumor-spheres were allowed to develop for 21 days (primary spheres). To generate secondary spheres, primary ones were disaggregated with trypsin and seeded again for 2 additional weeks. Images were captured with an Axio Vert microscope (A-Plan 5×/0.12 objective, Zeiss, Oberkochen, Germany), and evaluated with Carl Zeiss ZEN 2.3 SP1 (black) software. Quantification of spheres area was performed using ImageJ software, evaluating random quadruplicates.
4
Expansion of Cancer Stem Cells
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5000 cells/mL were seeded in non-adherent bacterial plates with CSC medium (DMEM-F12 without L-Glutamine nor Hepes (Biowest, Nuaillé, France) supplemented with 1% Penicillin/Streptomycin (Biowest, Nuaillé, France), B-27™ (40 mL/L, ThermoFisher Scientific, Waltham, MA, USA), human FGF-2 (0.01 µg/mL, Miltenyi Biotec, Bergisch Gladbach, Germany) and human EGF (0.02 µg/mL, Miltenyi Biotec, Bergisch Gladbach, Germany). Medium was renewed weekly by low speed centrifugation (5 min, 800 rpm) until primary spheres were obtained after 3 weeks. To generate secondary spheres, primary spheres were disaggregated through a 29G needle and seeded again at a density of 5000 cells/mL in 60 mm suspension culture dishes (Corning, Corning, NY, USA). For assays using recombinant human ADAMTS1 (rhATS1, 2197-AD, R&D), spheres were grown in 6-well ultralow attachment plates (Corning, Corning, NY, USA), using CSC medium supplemented with 1 µg/mL rhATS1. For assays using conditioned medium (CM), fresh medium was collected from 24 h cultured MUM-2B cells and supplemented with B-27, FGF-2 and EGF as described above. Images were captured with an Axio Vert microscope (A-Plan 5x/0.12 objective, Zeiss, Oberkochen, Germany), and evaluated with Carl Zeiss ZEN 2.3 SP1 (black) software (Oberkochen, Germany). Sphere volume was calculated as: sphere volume = (π × length × width2)/6.
5
Live-Cell FRAP Microscopy Imaging
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We performed live cell Fluorescence Recovery After Photobleaching (FRAP) on a Zeiss LSM 710 confocal microscope equipped with a 1.46 numerical aperture (NA) Plan-Apo × 100 oil-immersion lens (Carl Zeiss, Germany). We performed photobleaching using 75% laser power (488 nm wavelength of an argon laser). We imaged the photobleached region every 500 ms for 75 cycles, with laser power attenuated to 1.5% intensity. We measured the fluorescence intensity at the bleached spot, a control unbleached spot, and background. We analyzed the data using Zen 2.3 SP1 (black) software (Carl Zeiss).
6
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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