Click it edu alexa fluor 488 hcs assay

Manufactured by Thermo Fisher Scientific

Sourced in United Kingdom, United States

The Click-iT® EdU Alexa Fluor® 488 HCS Assay is a fluorescence-based cell proliferation assay that detects DNA synthesis in cells. It utilizes 5-ethynyl-2'-deoxyuridine (EdU), a nucleoside analog of thymidine, which is incorporated into newly synthesized DNA during the S-phase of the cell cycle. The incorporated EdU is then detected using a copper-catalyzed covalent reaction with a fluorescent Alexa Fluor® 488 dye.

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Lab products found in correlation

Leica sp8 confocal microscope, by Leica camera (2 mentions) Lsm 700, by Zeiss (2 mentions) Lsm 880, by Zeiss (2 mentions) Resazurin, by Merck Group (1 mentions) 5 ethynyl 2 deoxyuridine edu, by Thermo Fisher Scientific (1 mentions) Phalloidin 546, by Thermo Fisher Scientific (1 mentions) Calpeptin, by Bio-Techne (1 mentions) Axio imager z2 microscope, by Zeiss (1 mentions) Poly l lysine (pll), by Merck Group (1 mentions)

15 protocols using click it edu alexa fluor 488 hcs assay

Proliferation Measurement via EdU Labeling

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For proliferation testing, EdU labeling, which can incorporate
into the DNA of cells during replication, was performed. hMSCs were
plated on different substrate at a density of 1250 per cm² and allowed to recover overnight, followed by treatment with 1×
EdU solution. When the incubation up to 48 h, cells were fixed and
permeabilized with 4% PFA and 0.1% Triton X-100, respectively. Following
these processes, samples were treated according to the manufacturer’s
protocol of Click-iT EdU Alexa Fluor-488 HCS Assay (Thermo Fisher
Scientific). All images were collected by a Leica SP8 confocal microscope
(Leica, Germany) with filters for DAPI and Alexa Fluor-488. For quantification,
lower magnification (10× objective) fields were collected within
regions of interest.

Xie J., Bao M., Bruekers S.M, & Huck W.T. (2017). Collagen Gels with Different Fibrillar Microarchitectures Elicit Different Cellular Responses. ACS Applied Materials & Interfaces, 9(23), 19630-19637.

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Proliferating pHepaRG Cell Proliferation Assay

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Proliferating pHepaRG cells treated or not with GSK-126 and GKS-J4 for 72 h were fluorescent labeled (5 h) with the Click-iT® EdU Alexa Fluor® 488 HCS Assay (Thermofisher) as manufacturer’s instruction.

Pediconi N., Salerno D., Lupacchini L., Angrisani A., Peruzzi G., De Smaele E., Levrero M, & Belloni L. (2019). EZH2, JMJD3, and UTX epigenetically regulate hepatic plasticity inducing retro-differentiation and proliferation of liver cells. Cell Death & Disease, 10(7), 518.

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Quantifying Cell Proliferation in 3D Microniches

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Cell proliferation was determined by EdU labeling. hMSCs were seeded in 3D microniches for 2 and 9 days, followed by treatment with 1 × EdU solution. When the incubation was up to 3 and 10 days, cells were fixed and permeabilized with 4% PFA and 0.1% Triton X-100, respectively. Following these processes, samples were treated according to the manufacturer’s protocol of Click-iT EdU Alexa Fluor-488 HCS Assay (Thermo Fisher Scientific). All images were collected by a Leica SP8 confocal microscope (Leica, Germany) with filters for DAPI and Alexa Fluor-488. For quantification, lower magnification (10× objective) fields were collected within regions of interest.

Bao M., Xie J., Piruska A, & Huck W.T. (2017). 3D microniches reveal the importance of cell size and shape. Nature Communications, 8, 1962.

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Cell Cycle Analysis with CDK7i Compounds

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Cells were plated in triplicate in 96-well plates. After 24 hours, cells were starved in FBS-free medium for 24 hours for cell cycle synchronization and then treated with the CDK7i SY-1365 or samuraciclib, palbociclib, or DMSO for 48 hours in their growth media. The cell cycle assay was performed using the Click-iT EdU Alexa Fluor 488 HCS Assay (Thermo Fisher Scientific) according to the manufacturer's instructions. Newly synthesized DNA was labeled with EdU (5-ethynyl-2′-deoxyuridine), positive cells were counted, and the cell cycle phases were estimated using the Celigo image cytometer.

Guarducci C., Nardone A., Russo D., Nagy Z., Heraud C., Grinshpun A., Zhang Q., Freelander A., Leventhal M.J., Feit A., Cohen Feit G., Feiglin A., Liu W., Hermida-Prado F., Kesten N., Ma W., De Angelis C., Morlando A., O'Donnell M., Naumenko S., Huang S., Nguyen Q.D., Huang Y., Malorni L., Bergholz J.S., Zhao J.J., Fraenkel E., Lim E., Schiff R., Shapiro G.I, & Jeselsohn R. (2024). Selective CDK7 Inhibition Suppresses Cell Cycle Progression and MYC Signaling While Enhancing Apoptosis in Therapy-resistant Estrogen Receptor–positive Breast Cancer. Clinical Cancer Research, 30(9), 1889-1905.

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Cell Cycle Synchronization and Drug Response

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Cells were seeded into 96-well plates at 3000 cells/well density. The plated cells were starved in FBS-free culturing medium for 16 h for cell cycle synchronization, followed by treatment with vehicle, fulvestrant, 5FU or fulvestrant in combination with 5FU. After 24 h of drug exposure, cell cycle assay was performed using the Click-iT™ EdU Alexa Fluor™ 488 HCS Assay (ThermoFisher Scientific) according to the manufacturer's instructions. Newly synthesized DNA was labeled with EdU (5-ethynyl-2'-deoxyuridine), positive cells were counted, and the cell cycle phases were estimated using the Celigo image Cytometer.

Grinshpun A., Russo D., Ma W., Verma A., Hermida-Prado F., Sherman S., Gaglia G., Kabraji S., Kirkner G., Hughes M.E., Lin N.U., Sandusky Z., Nardone A., Guarducci C., Nguyen Q.D., Santagata S., Nagy Z, & Jeselsohn R. (2024). Pure estrogen receptor antagonists potentiate capecitabine activity in ESR1-mutant breast cancer. NPJ breast cancer, 10(1).

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Cell Proliferation, Migration, and Metabolic Assays

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For analysis of cell proliferation, 20 μM EdU was added for 2 h, and the cells were fixed and analyzed by Click-iT™ EdU Alexa Fluor™ 488 HCS Assay (Thermo Fisher Scientific, #C10350) as described [16] . For analysis of cell migration, the monolayer of hCFs was wounded by scratching with a sterile plastic 200 μL micropipette tip, washed with PBS, and photographed as described [18] . After incubation for 7 h, cells were fixed and photographed again using a low-magnification phasecontrast microscope. The extent of migration into the wound area was analyzed by using Adobe Photoshop. Cell metabolic activity was determined by incubating cells with 10 μM resazurin (Sigma, #R7017) for 1 h as previously described [16] , and measuring with excitation at 544 nm and emission at 590 nm by a multi-plate reader (VarioskanFlash 4.00.53).

Lin R., Rahtu-Korpela L., Szabo Z., Kemppi A., Skarp S., Kiviniemi A.M., Lepojärvi E.S., Halmetoja E., Kilpiö T., Porvari K., Pakanen L., Tolva J., Paakkanen R., Segersvärd H., Tikkanen I., Laine M., Sinisalo J., Lakkisto P., Huikuri H., Magga J., Junttila J, & Kerkelä R. (2022). MiR-185-5p regulates the development of myocardial fibrosis. Journal of molecular and cellular cardiology, 165.

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Evaluating DNA Synthesis in FLS Cells

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FLS were cultured in the presence of Smac 060 or 066 (20 μM, overnight) or in tissue medium alone (CTRL). DNA synthesis was evaluated by adding 10μ M 5-ethynyl-2-deoxyuridine (EdU) labelled with Alexa Fluor 488 dye (Click-iT® EdU Alexa Fluor® 488 HCS Assay, Invitrogen, Carlsbad, CA). After 24-h incubations, cells were fixed and permeabilised. We evaluated EdU incorporated into newly synthesised DNA by detecting the fluorescent Alexa Fluor 488 label. Analysis was performed with fluorescence confocal microscopy.

Lattuada D., Casnici C., Crotta K., Seneci P.F., Corradini C., Truzzi M., Ingegnoli F, & Marelli O. (2014). Proapoptotic Activity of a Monomeric Smac Mimetic on Human Fibroblast-Like Synoviocytes from Patients with Rheumatoid Arthritis. Inflammation, 38(1), 102-109.

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Cell Proliferation Assay with EdU Labeling

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For assessing cell proliferation, 10 μM EdU was added to the culture medium after 2 weeks of transduction and maintained throughout the culture for an additional 2 weeks. Cells were fixed with 4% PFA for 15 min, permeabilized, and incubated with anti-cTNT antibodies. Cells were then incubated with secondary antibodies conjugated with Alexa Fluor 546 (for immunocytochemistry) or 647 (for FACS) and then incubated with the EdU reaction cocktail following the manufacturer’s instructions (Click-iT EdU Alexa Fluor 488 HCS Assay; Invitrogen, C10350).

Yamakawa H., Muraoka N., Miyamoto K., Sadahiro T., Isomi M., Haginiwa S., Kojima H., Umei T., Akiyama M., Kuishi Y., Kurokawa J., Furukawa T., Fukuda K, & Ieda M. (2015). Fibroblast Growth Factors and Vascular Endothelial Growth Factor Promote Cardiac Reprogramming under Defined Conditions. Stem Cell Reports, 5(6), 1128-1142.

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Quantifying Plant Cell Proliferation

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11-day-old seedlings were incubated with 5-ethynyl-2′-deoxyuridine (EdU) (1 μM) (Cat#C10350, Invitrogen) for 30 min in liquid 1/2 MS medium. Then, samples were fixed with 1× PBS solution containing 4% formaldehyde and 0.1% Triton x-100 for 30 min at 4 °C. After fixation, samples were washed with 1× PBS 3 times with rotation for 5 min at RT, then conjugated to Alexa Fluor 488 with the Click-iT EdU Alexa Fluor 488 HCS Assay (Cat#C10350, Invitrogen) for 30 min in the dark at RT. Samples were then washed with 1× PBS 3 times with rotation for 10 min at RT and imaged using a stereomicroscope (Leica DM6B).

He L., Huang H., Bradai M., Zhao C., You Y., Ma J., Zhao L., Lozano-Durán R, & Zhu J.K. (2022). DNA methylation-free Arabidopsis reveals crucial roles of DNA methylation in regulating gene expression and development. Nature Communications, 13, 1335.

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Detecting S-Phase Cells in Arabidopsis Seedlings

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5-ethynyl-2′-deoxyuridine (EdU) staining using an Invitrogen Click-iT^™ EdU Alexa Fluor^™ 488 HCS Assay (C10350) was performed based on Kotokany et al. (2010) [20 (link)] to detect S phase cells. Seeds were grown in MS media vertically for 3 days. Seedlings were collected in MS solution containing 1μM Edu and incubated at 22°C for 30 minutes. Samples were fixed in 4%(w/v) formaldehyde solution in phosphate-buffered saline (PBS) with 0.1% Triton X-100 for 30min, and washed three times with PBS each for 5 minutes. The samples were incubated in Edu detection cocktail solution at room temperature for 30 minutes in the dark, and washed with the Click-iT^® rinse buffer and then three times with PBS. The photos were taken using confocal microscopy (LSM700, Zeiss).

Frost J.M., Lee J., Hsieh P.H., Lin S.J., Min Y., Bauer M., Runkel A.M., Cho H.T., Hsieh T.F., Fischer R.L, & Choi Y. (2023). H2A.X promotes endosperm-specific DNA methylation in Arabidopsis thaliana. Research Square.

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