hPSC-RPE cells were cultured on Transwell membranes (0.33 cm2, Millipore) coated with different substrates. Supernatants from both the hPSC-RPE apical and basal sides (meaning upper and lower compartments of the transwell, respectively) were collected 60 h after the medium was changed. PEDF secretion levels were measured in triplicates for each condition with commercially available human PEDF ELISA Kits (BioVendor RD191114200R) were used, in accordance with the manufacturer’s instructions, after 60 days of culture. The optical density Sreadings were measured using SpectraMax 250 Microplate Reader (Molecular Devices). Results are presented as mean ± SEM.
Human pedf elisa kit
Manufactured by BioVendor
Sourced in Canada
The Human PEDF ELISA kit is a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) designed for the measurement of PEDF (Pigment Epithelium-Derived Factor) levels in human samples. It is a laboratory tool used to detect and quantify PEDF protein concentration in various biological matrices.
Automatically generated - may contain errors
Lab products found in correlation
Millicell voltohmmeter, by Merck Group (2 mentions)
Lsm 700 800 confocal microscope, by Zeiss (2 mentions)
Zen 2.3 sp1 black software, by Zeiss (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Spectramax 250 microplate reader, by Molecular Devices (1 mentions)
Transwell membrane, by Merck Group (1 mentions)
Prolong gold mounting medium, by Thermo Fisher Scientific (1 mentions)
Vegf human elisa kit, by Thermo Fisher Scientific (1 mentions)
5 protocols using human pedf elisa kit
1
PEDF Secretion in hPSC-RPE Cells
2
VEGF and PEDF Quantification Protocols
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
VEGF and PEDF were performed according to the manufacturer’s protocol of the VEGF Human ELISA Kits (Invitrogen, Thermo Fisher Scientific Inc., Waltham, MA) and Human PEDF ELISA Kits (BioVendor, Brno, Czech Republic), respectively.
3
Characterization of Human ESC-Derived RPE
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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 ).
4
Preserving hiPSC-RPE Cell Secretion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
VEGF and PEDF secretion of hiPSC-RPE cells was examined in tubes at each temperature and in a 6-well CELLstart-coated plate at 37 °C. We added a plate culture condition to compare between VEGF and PEDF secretion in different vessels at the same temperature. Immunoassay kits were used to examine secretion of VEGF (VEGF Human ELISA Kit, Life Technologies, Carlsbad, CA) and PEDF (Human PEDF ELISA Kit, Biovendor, Brno, Czech Republic). Total protein content in the supernatant was determined as the volume of the medium differs in tube and plate cultures. For recovery cultures, fresh medium was added, and after 24 hours, the protein content of the supernatants was examined. At each time point, all media were collected from tubes and wells and kept in frozen storage until assayed. In recovery cultures, VEGF and PEDF secretion of hiPSC-RPE cells preserved at each temperature was examined. After preservation, hiPSC-RPE cells were seeded into 24-well CELLstart-coated plates and cultured at 37 °C for 13 days. Supernatants were collected after 24 hours, stored for testing, and replaced with fresh medium.
5
Characterization of Human ESC-RPE Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!