Cells were treated under experimental conditions for up to 96 hours before collection. For the senescence assay, CellEvent Senescence Green Flow Cytometry Assay (Thermo Fisher Scientific) was performed according to the manufacturer's instructions, with staining at 10 μmol/L probe for 90 minutes at 37°C. For the proliferation assay, Baseclick EdU Flow Cytometry Kit (Sigma-Aldrich) was performed according to the manufacturer's instructions, with incubation at 10 μmol/L EdU for 90 minutes. Data were acquired on the BD LSRFortessa (BD Biosciences; OSUCCC FLOW Cytometry Shared Resource) and analyzed using FlowJo (FlowJo, LLC).
Baseclick edu flow cytometry kit
Manufactured by Merck Group
Sourced in Ireland
The Baseclick EdU Flow Cytometry Kit is a product designed for use in flow cytometry applications. It enables the detection and analysis of cell proliferation through the incorporation of EdU (5-ethynyl-2'-deoxyuridine) into newly synthesized DNA. The kit provides the necessary reagents and protocols for this specific purpose.
Automatically generated - may contain errors
4 protocols using baseclick edu flow cytometry kit
1
Senescence and Proliferation Assays
2
EdU Flow Cytometry for Cell Proliferation
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The detection of cell proliferation and DNA content was performed using the baseclick EdU Flow Cytometry Kit (baseclick, Sigma-Aldrich, Arklow, Ireland) according to the manufacturer’s instructions. In brief, after 72 h exposure time, cells were incubated with 10 µM EdU (5-ethynyl-2′-deoxyuridine) for 2 h. Then, cells were harvested, centrifuged at 500× g for 5 min, washed with 1% BSA in PBS and fixed with the fixative solution. After a second wash with 1% BSA in PBS, cells were incubated with saponin-based permeabilization buffer in PBS before starting the click reaction. Cells were then washed with saponin-based permeabilization buffer and wash reagent, and incubated with a 10 µg/mL Propidium Iodide (PI)/100 µg/mL RNase solution in PBS (PI/RNase, Sigma-Aldrich, Arklow, Ireland) for 15 min at 4 °C. EdU and PI fluorescence was then measured using the CytoFLEX Flow Cytometer with a blue laser (488 nm) using FL-1 and FL-2 detectors. Data were analysed using CytExpert version 2.4.0.28 software (Beckman Coulter Inc., Indianapolis, IN, USA). For each assay, data of at least 10,000 events were collected.
3
Cellular Senescence and Proliferation Assays
4
Captan-induced Cell Proliferation Assay
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Two hours after captan treatment (10 µM), CHO cell lines were incubated with EdU (5 µM) for 15 min. Cells were collected by trypsinization and incorporated EdU was detected using the baseclick EdU flow cytometry kit (Sigma, BCK-FC488) according to the manufacturer's instructions. Cells were incubated in PBS containing DAPI (1 µg/ml) for 15 min before samples were processed using flow cytometry (MACSQuant, Miltenyi Biotec) . At least 10,000 events were analysed per sample using FlowJo software.
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