EdU assay kit (Abcam, ab219801) was used to assess the proliferation of enteroids incubating with B longum according to the manufacturer’s instructions. In brief, 10 μM EdU was added to the medium 1 hour after co-culture and incubated for additional 3 hours. The matrigel was dissociated with Cell Recovery Solution (Corning, #354253) for 15 minutes at 4 °C, and afterward enteroids were digested in 0.25% Trypsin + 5% EDTA in PBS for 30 minutes in a 37°C water bath. Then, 10% BSA-PBS was added to stop the digestion and cells were centrifuged at 400 × g for 5 minutes at 4 °C and re-suspended in 3% BSA-PBS. After filtration using a 40 μm aperture strainer, cells were fixed with 4% PFA, permeated and incubated with EdU Reaction mix. Then cells were washed twice and re-suspended in 200 ~ 300 µL PBS for analyzing by Moflo XDP (Beckman Coulter) at Ex/Em = 491/520 nm.
Edu assay kit
Manufactured by Abcam
Sourced in United States, United Kingdom
The EdU assay kit is a tool for detecting and quantifying cell proliferation. It utilizes the incorporation of the thymidine analog EdU (5-ethynyl-2'-deoxyuridine) into newly synthesized DNA, allowing for the identification of proliferating cells.
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8 protocols using edu assay kit
1
Quantifying Enteroid Proliferation with EdU Assay
2
EdU Assay for Cell Proliferation
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An EdU assay kit (Abcam, United States) was used to detect DNA synthesis and cell proliferation. We seeded 1 × 104 SGC-7901 or BGC-823 cells in a 96-well plate overnight and then added Edu solution (25 μM) into the wells and incubated them for 24 h. Then, 4% paraformaldehyde was applied at RT for 30 min to fix the cells. Triton X-100 (0.5%) was added for 10 min to permeabilize the cells, and then Apollo reaction solution (200 μL) was added to stain the EdU for 30 min and Hoechst 33342 (200 μL) was added to stain the nuclei. Finally, we visualized the cells under a fluorescence microscope (IX81, Olympus, Japan) to observe DNA synthesis and cell proliferation.
3
Quantifying Cell Proliferation by EdU Assay
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Cell proliferation was measured by EdU assay kit (Abcam) as per manufacturer’s instructions. Cells were seeded in 6-well plates on a coverslip at a density of 5 × 10³ cells per well post-transfection. Following this, the cells were treated with 20 μM EdU labelling medium and were cultured at 37°C under 5% CO₂ for 4 hours. The cultured cells were fixed for 30 min with 4% paraformaldehyde (pH= 7.4). Cells were then washed and treated with permeabilization buffer for 20 min at RT. EdU reaction cocktail (200 μL) was then added to react with EdU for 30 min followed by addition of Hoechst 33342 (100 μL) to the cells. Fluorescent microscopy was utilized to analyze the percentage of EdU positive cells in five random fields per well.
4
Cell Proliferation Assay Using EdU
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The infected ZBTB7A-Huh7 cells were plated in 24-well plate. EdU Assay Kit (Abcam, ab219801) was applied following the protocol. Briefly, EdU solution was diluted with DMEM media at 20μM and added into cells to be stained for 4 h. The cells were washed with PBS and then incubated with fixative solution and permeabilization buffer separately for 15 min. The reaction buffer containing iFluor 488 dye was added and incubated for 30 min. The samples were analyzed by fluorescence microscope.
5
Measuring Cell Proliferation with EdU Assay
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An EdU assay kit (product code ab219801; Abcam) was used to assess cell proliferation. Following cell dissociation, the cells were diluted to 1×106 cells/ml with culture medium and labeled with EdU solution for 4 h. The cells were then fixed with 4% paraformaldehyde for 15 min at room temperature, permeabilized in permeabilization buffer and incubated with EdU reaction mix at room temperature for 30 min. Finally, the number and proportion of EdU-incorporated cells were analyzed on a flow cytometer (FACSVerse; BD Biosciences) at Ex/Em=491/520 nm. The data were analyzed using Cell Quest software v5.1 (BD Biosciences).
6
Measuring Cell Proliferation with EdU Assay
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Cells were incubated for 16 h with EdU after exposure to 10Gy. Then the cells were processed as mentioned in the EdU Assay Kit (Abcam, ab219801).
7
Proliferation Rate of CMSCs
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The proliferation rate of CMSCs was tested using an EdU assay kit (Abcam), an index of DNA replication. CMSCs were seeded with a density of 2.5 × 104/cm2 cells, and after 24 h, they were incubated with medium, supplemented with 40 μM EdU, at 37 °C for 4 h. Subsequently, CMSCs were detached, fixed with the fixative solution (4% formaldehyde-based) and permeabilized with the permeabilization buffer (Triton X-100-based). For EdU detection, cells were incubated with iFluor 488 azide and measured with the Gallios flow cytometer platform by using Kaluza 1.1 acquisition software (Beckman Coulter, Brea, CA, USA). Data from 5000 events per sample were collected and examined with Kaluza 1.3 analysis software.
8
Cell Proliferation Assay with EdU
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EdU Assay Kit (Abcam, Cambridge, UK) was obtained, and 2× EdU solution was added in culture medium for another 4 h in the dark; then, these cells were fixed in 1× Working Fixative Solution for 15 min, washed with the Wash Buffer twice; cells were permea-bilized after 1× Permeabilization Buffer incubation for 20 min. Eventually, cells were processed in the EdU Reaction Mix for 30 min without sunlight, followed by DAPI working solution. The stained cells were imaged under a fluorescence microscope (Olympus IX73; Olympus, Tokyo, Japan) equipped with filter for Ex/Em = 491/520 nm, and the percentage of EdU-positive cells in DAPI-positive cells was determined in five randomly selected fields (100×).
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