The DB or SU-DHL-4 cells were stained with 10 µM EdU reagent (Nanjing KeyGen Biotech. Co., Ltd.) at 37°C for 2 h, and fixed with 4% paraformaldehyde for 15 min. Subsequently, all cells were washed with PBS containing 3% bovine serum albumin (BSA), incubated with 0.5% Triton X-100 for 20 min and incubated with Click-iT reagent (Nanjing KeyGen Biotech. Co., Ltd.) at room temperature for 30 min in the dark. Finally, the cells were stained with DAPI reagent (Beijing Solarbio Science & Technology Co., Ltd.) at room temperature for 5 min, and photographed at a ×400 magnification.
Edu reagent
Manufactured by Keygen Biotech
Sourced in China
The EdU reagent is a chemical compound used in biological research. It serves as a tool for labeling and detecting proliferating cells. The core function of the EdU reagent is to enable the visualization and quantification of cell proliferation in various cell and tissue samples.
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4 protocols using edu reagent
1
Cell Proliferation Assay using EdU
2
EdU Incorporation in NSCLC Cells
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NSCLC cells were stimulated with 100 μl medium plus 20 μM EdU reagent (keyGEN BioTECH). After 2‐h incubation, NSCLC cells were immobilized via 4% paraformaldehyde stationary liquid (Sangon Biotech) and mixed with 0.5% surfactant Triton‐X‐100 solution (Sangon Biotech). Cell nuclei was dyed using 4, 6‐diamino‐2‐phenylindole dye liquor (DAPI; Sigma). The fluorescence signals were analyzed via a fluorescence microscope (Leica).
3
Meningioma Cell Viability and Proliferation
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Cell viability was assessed using the CCK-8 method. Briefly, meningioma cells were plated in 96-well plates for 24–72 h, followed by the addition of 10 μL of CCK-8 reagent (MCE) and incubation at 37°C for 2 h. The absorbance at 450 nm was measured using a microplate reader. For EdU staining, cells or 3D tumorspheres were incubated with 10 μM EdU reagent (KeyGEN) for 2 h; they were subsequently trypsinized into cell suspensions and fixed using 4% paraformaldehyde. According to the instructions of the reagent's manufacturer, EdU-positive cells were fluorescently labeled with FITC, and the positive-cell ratio was determined using flow cytometry.
4
Cell Proliferation Assays in 96-well Plates
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After cell culture in 96-well plates, 1 h of incubation was performed using cell counting Kit-8 (CCK-8) reagent (Beyotime, Nantong, China). Then, a TECAN infinite M200 Multimode microplate reader (Tecan, Mechelen, Belgium) was employed to record the absorbance at 450 nm.
For a 5-ethynyl-2′-deoxyuridine (EdU) assay, 2 h of cell incubation was conducted in EdU reagent (Keygen, Nanjing, China). Before EdU staining following the manufacturer’s recommendations, 15 min of cell fixation was performed in 4% paraformaldehyde (Beyotime, Nantong, China).
For a 5-ethynyl-2′-deoxyuridine (EdU) assay, 2 h of cell incubation was conducted in EdU reagent (Keygen, Nanjing, China). Before EdU staining following the manufacturer’s recommendations, 15 min of cell fixation was performed in 4% paraformaldehyde (Beyotime, Nantong, China).
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