For BrdU incorporation analysis, mice intraperitoneally injected with 0.1mg/kg BrdU and maintained on 0.5mg/ml BrdU water for 5 days before the analysis. The frequency of BrdU+ cells was determined by flow cytometry using an APC BrdU Flow Kit (BD Biosciences) per manufacturer’s instructions. To measure γH2AX levels, cells were stained for SLAM markers, fixed and permeabilized (BD Biosciences BrdU Flow Kit) and stained with anti-γH2AX (Biolegend, clone 2F3), followed by flow cytometry. Annexin V staining was performed using Annexin V Apoptosis Detection Kit APC (eBioscience) per manufacturer’s instructions. ROS levels were measured by DCFDA staining (29–79-dichlorofluorescein diacetate, ThermoFisher Scientific). After antibody staining, cells were incubated with 10uM DCFDA for 15 minutes at 37°C, followed by flow-cytometry.
29 79 dichlorofluorescein diacetate
Manufactured by Thermo Fisher Scientific
29–79-dichlorofluorescein diacetate is a fluorogenic probe used for the detection and measurement of intracellular esterase activity. It is a non-fluorescent, membrane-permeable compound that can be hydrolyzed by cellular esterases to produce a fluorescent product.
Automatically generated - may contain errors
2 protocols using 29 79 dichlorofluorescein diacetate
1
Multimodal Analysis of Cell Proliferation and Stress
2
Multimodal Analysis of Cell Proliferation and Stress
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For BrdU incorporation analysis, mice intraperitoneally injected with 0.1mg/kg BrdU and maintained on 0.5mg/ml BrdU water for 5 days before the analysis. The frequency of BrdU+ cells was determined by flow cytometry using an APC BrdU Flow Kit (BD Biosciences) per manufacturer’s instructions. To measure γH2AX levels, cells were stained for SLAM markers, fixed and permeabilized (BD Biosciences BrdU Flow Kit) and stained with anti-γH2AX (Biolegend, clone 2F3), followed by flow cytometry. Annexin V staining was performed using Annexin V Apoptosis Detection Kit APC (eBioscience) per manufacturer’s instructions. ROS levels were measured by DCFDA staining (29–79-dichlorofluorescein diacetate, ThermoFisher Scientific). After antibody staining, cells were incubated with 10uM DCFDA for 15 minutes at 37°C, followed by flow-cytometry.
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