Cell proliferation was analysed using a Click-iT™ EdU Proliferation Assay for Microplates (Thermo Fisher Scientific, Cat# C10499). Briefly, the nucleoside analog EdU (5-ethynyl-2'-deoxyuridine) was added to live cells for 4 h. Then the cells were fixed and incubated with horseradish peroxidase (HRP), which becomes ligated to the DNA-incorporated EdU moiety by using click chemistry. Thereafter, AmplexTM UltraRed, a reagent that is converted to a fluorescent product in presence of HRP, was added and the fluorescence was measured on a microplate reader at an excitation of 568 nm and an emission of 585 nm.
Click it edu proliferation assay for microplates
Manufactured by Thermo Fisher Scientific
The Click-iT™ EdU Proliferation Assay for Microplates is a tool used to quantify cell proliferation. It allows for the detection and measurement of newly synthesized DNA in proliferating cells through the incorporation of the nucleoside analog EdU (5-ethynyl-2'-deoxyuridine).
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7 protocols using click it edu proliferation assay for microplates
1
Cell Proliferation Measurement by EdU Assay
2
Proliferation and Insulin Secretion of EndoC-βH1 Cells
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The EndoC-βH1 human beta cell line was cultured consistent with conditions described by Tsonkova et al [11 (link)]. The EndoC-βH1 was cultured in DMEM low glucose (1g/L), 2% Albumin from bovine serum fraction V, 50 μM 2-mercaptoethanol, 10 mM nicotinamide, 5.5 μg/ml transferrin, 6.7 ng/ml sodium selenite and Penicillin (100 units/ml) / Streptomycin (100 μg/ml) and maintained in sub confluent densities. EndoC-βH1 cells were seeded at 2 ×105 cells per well in a cell culture treaded 96-well plate and allowed to adhere for 24 hours prior to starting of the experiments. The next day, seeding media was exchanged for fresh media containing 10% human serum from pregnant or non-pregnant donors together with EdU (Click-iT™ EdU Proliferation Assay for Microplates, ThermoFisher Scientific) and cultured for 1 week. At takedown, EdU was detected following the kit manufacturer’s protocol and fluorescence was measured with a microplate reader (Molecular Dives Spectramax M5) at 568/585 nm. Secreted insulin in cell culture supernatant was measured by human Insulin Chemiluminescence ELISA kit (ALPCO).
3
Cell Proliferation Assay with EdU
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10,000 hTERT‐HPNE or HPDE‐H6c7 cells were seeded in 96‐well plates and allowed to attach overnight. The next day, cells were treated as indicated in the figures. Proliferation was measured using the Click‐iT EdU Proliferation Assay for Microplates (ThermoFisher, #C10499). At the end of the treatment time point, EdU at a final concentration of 10 μM per well was added to the cells. After an additional 18h of incubation, cells were fixed and analysed per manufacturers protocol.
4
Primary Human Hepatocyte Culture and Proliferation
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Cryopreserved primary human hepatocytes (Lonza #HUCPG) were cultured according to the supplier’s instructions. Hepatocytes were thawed at 37°C and placed in warmed thawing medium (Lonza # MCHT50P), resuspended in Hepatocyte Plating Medium (Lonza #MP100), plated in collagen-coated 96 well plates at 50,000 cells/well, and cultured at 37°C and 5% CO2. Culture wells were precoated with 140 μg/ml Collagen I solution (Roche #11179179001) for 2 hrs at 37°C. The initial plating medium was exchanged for fresh plating medium after 1h. Plating media was then exchanged again for Hepatocyte Culture Medium (Lonza #CC-4182) after 24 h, and then daily for the next 3 days. Once primary human hepatocyte monolayers were established, 10% pooled human serum from pregnant or non-pregnant donors was added together with EdU (Click-iT™ EdU Proliferation Assay for Microplates, ThermoFisher Scientific) and cultured for one week. At takedown, EdU was detected following the manufacturer’s protocol and fluorescence was measured with a microplate reader (Molecular Dives Spectramax M5) at 568/585 nm.
5
BMVECs Proliferation Assay with EdU
6
Cell Proliferation Quantification by EdU Assay
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Proliferation was assessed using the Click-iT EdU Proliferation Assay for Microplates (C10499, Invitrogen). Briefly, cells were plated at 48% confluency in 96 well black microplate (CLS3603, Corning, MERCK), labeled with 10 µM EdU for 24 h, then fixed and clicked according to the manufacturer’s instructions. Fluorescence was analyzed with a Mithras LB 940 reader (Berthold technologies, Bad Wildbad, Germany) and MikroWin 2010 software (Berthold technologies) using excitation filter 560 nm and emission filter 590 nm.
7
Assessing Cell Proliferation with EdU
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N/TERT2G cells were plated in 96-well plates at 28,000 cells/well. After 24 hours, 10 μM 5-ethynyl-2′-deoxyuridine was added to each well and incubated for 6 or 24 hours. Proliferative capacity was assessed by the Click-iT EdU Proliferation Assay for Microplates (C10499, Invitrogen, Waltham, MA), and the protocol was followed as written in the product information sheet.
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