Cell-proliferation assays were conducted by labeling genomic DNA with EdU (a DNA precursor molecule that is incorporated during DNA replication) and measuring such incorporation using the Click-iTTM EdU Flow Cytometry Assay Kit (Invitrogen, CA, USA) 35 (link).
Click ittm edu flow cytometry assay kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Click-iTTM EdU Flow Cytometry Assay Kit is a reagent-based system designed to detect and analyze cellular proliferation using flow cytometry. The kit utilizes the incorporation of the thymidine analog EdU (5-ethynyl-2'-deoxyuridine) into newly synthesized DNA during the S phase of the cell cycle. The incorporated EdU can then be detected using a fluorescent azide dye, allowing for the quantification of proliferating cells.
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4 protocols using click ittm edu flow cytometry assay kit
1
Cell Proliferation Assay with EdU
2
Cell Cycle Analysis using Click-iT EdU Assay
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Cell cycle analysis was performed using Click-iTTM EdU Flow Cytometry Assay Kit by Invitrogen according to the manufacturer’s protocol. In brief, EdU was added to the cells as per dilution 1:1000, after 2 h cells were harvested and stained for 3t3-feeder cells. Cells were then fixed, permeabilized, incubated with Click-iT reaction cocktail and, at last, stained with FxCycleTMViolet. Stained cells were analyzed with BD FACSCanto II, BD FACSDiva Software v6,1,3 and FlowJo v10.
3
EdU-Based Cell Proliferation Analysis
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4
Proliferation Dynamics of Endothelial Cells and Pericytes
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The endothelial cells and the pericytes (2 × 107 cells) were incubated with Epo B (10 ρM, 100 ρM) for 24 h. 5-Ethynyl-2′-deoxyuridine (EDU; Click-iTTM EDU Flow Cytometry Assay Kit, InvitrogenTM, OR USA) was added to endothelial cells and pericytes at a final concentration of 50 μM and 40 μM, and the cells were incubated for further 2 h and 6 h, respectively. The cells were washed three times in PBS and incubated under the OGD condition for 60 min. Then cells (2 × 107) were harvested (1000g, 5 min), washed (1% BSA in PBS, 3 ml), fixed (Click-iTTM fixative, 100 μL), permeabilized (10% Click-iTTM saponin-based permeabilization and wash reagent in PBS), and incubated with Click-iTTM Plus reaction cocktail (10 μL copper protectant, 2.5 μL fluorescence dye picolylazide, 50 μL reaction buffer additive, and 435 μL PBS) according to the manufacturer’s instructions for the Click-iTTM EDU Flow Cytometry Assay Kit. Flow cytometry was performed to analyze cell proliferation.
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