Human renal VSMCs at passages 3–8 were seeded onto poly-l-lysine coated coverslips placed in 12 mm wells at a density of 20,000 cells/well. Cells were maintained in control medium for 24 hours and synchronized in serum free (SF) medium during 48 hours. Then, PDGF (20 ng/ml) was added for an additional 24 hours period. Tungstate and/or the BK channel blocker IbTX were added one hour before and remained present during PDGF stimulation. The percentage of cells at the S phase was quantified using EdU (5-ethynyl-2´-deoxyuridine) incorporation for another 6 hours with a commercial kit (Click-iT EdU Imaging Cell Proliferation Assay, Invitrogen). Finally, cells were incubated with Hoechst before mounting with Vectashield (Vector Laboratories Inc.,Burlingame, CA). EdU incorporation was visualized with an immunofluorescence microscopy (Nikon) at the corresponding wavelength depending on the Alexa Fluor used and was expressed as the percentage of the total cell number stained with Hoechst. In each experiment, this percentage was an average of 10 to 20 high power fields per coverslip. Triplicates were made for each condition.
Click it edu imaging cell proliferation assay
Manufactured by Thermo Fisher Scientific
The Click-iT EdU Imaging Cell Proliferation Assay is a fluorescence-based kit for detecting and quantifying cell proliferation in cultured cells. It uses a nucleoside analog of thymidine, EdU (5-ethynyl-2'-deoxyuridine), which is incorporated into DNA during active DNA synthesis. The incorporated EdU can then be detected using a copper-catalyzed click reaction with a fluorescent azide.
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2 protocols using click it edu imaging cell proliferation assay
1
Quantifying PDGF-induced Renal VSMC Proliferation
2
Quantifying VSMC Proliferation with EdU
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The percentage of cells at the S phase was quantified using EdU (5-ethynyl-2´-deoxyuridine) incorporation for 6h with a commercial kit (Click-iT® EdU Imaging Cell Proliferation Assay, Invitrogen) as described [11] (link). Briefly, 25,000 VSMCs were seeded onto fibronectin-coated coverslips placed in 12 mm wells with control media (10% FBS). Next day, media was replaced with serum-free (SF) media, and proliferation was induced by addition of PDGF (20 ng/ml), alone or in the presence of PAP-1 (100nM) or ELR-containing-PAP-during 24 h. Triplicate samples were used, and controls were included in all experiments. Proliferation was determined as the percentage of EdU positive cells (EdU+) from the total cell number stained with Hoechst, obtained from the average of 4 randomly selected images per coverslip, captured using the 4X objective in a Nikon Eclipse 90i microscope. ImageJ (Fiji) software was used to analyze the images in a blind manner.
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