Apoptotic cell death was detected by flow cytometry using Annexin V and 7-amino-actinomycin (7-AAD) staining. Cells were harvested and resuspended in Annexin V-binding buffer containing 10% Annexin V-FITC and 10% 7-AAD staining solution (Thermo Fisher Scientific). After an incubation time of 15 min at 4 °C, stained cells were analyzed by flow cytometry.
7 aad staining solution
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 7-AAD (7-Aminoactinomycin D) staining solution is a fluorescent dye used in flow cytometry applications to detect cell viability. It binds to DNA and emits a fluorescent signal when excited, allowing for the identification of dead or compromised cells within a sample.
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5 protocols using 7 aad staining solution
1
Annexin V and 7-AAD Apoptosis Assay
2
Annexin V and 7-AAD flow cytometry
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Apoptotic cell death was detected by flow cytometry using Annexin V and 7-amino-actinomycin (7-AAD) staining. Cells were harvested and resuspended in Annexin V-binding buffer containing 10% Annexin V-FITC and 10% 7-AAD staining solution (Thermo Fisher Scientific). After an incubation time of 15 min at 4°C, stained cells were analyzed by flow cytometry.
3
Annexin V-APC and 7-AAD Cell Apoptosis Assay
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Approximately 5 × 105 of A549 and SPCA-1 cells were harvested and suspended in 500 μl of binding buffer. Then, 5 μl of Annexin V-APC and 5 μl of 7-AAD staining solution (Invitrogen, Carlsbad, CA, USA) were added, and the cells were incubated at 37°C for 15 min in the dark. Flow cytometry analysis was used (BD-FACSCalibur, Franklin Lakes, NJ, USA) to detect red fluorescence of cells. Finally, the results were analyzed using FlowJo software, version 10.0 (FlowJo, Ashland, OR, USA).
4
DNA Damage and Cell Cycle Analysis
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WT Sol8 and GneKO cells were detached from the culture dish using trypsin, washed with PBS, counted and were then fixed and permeabilized using ice cold 70% ethanol for at least 2 h at −20°C. After PBS washing, the cells were stained for 30 min on ice with the relevant antibody/staining solution (1:50 Alexa Fluor 488 conjugated anti γ-H2AX #BLG-613406, BioLegend; 1:50 rabbit anti-Ki67 #275R-14 Cell Marque; 1:25 7AAD staining solution #00-6993-50 Invitrogen). Following washing, the Ki67 stained cells were incubated in the dark for 30 min on ice with donkey anti rabbit Alexa Fluor 488 (1:150, #711-546-152, Jackson, United States).
5
Cell Cycle Analysis with Flow Cytometry
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As described in a previous study54 (link), SiHa, CaSki and HeLa cells were harvested in PBS and fixed in ice-cold 70% ethanol, which was added with a Pasteur pipette with vortexing. Then, the cells were centrifuged at approximately 2,000 rpm for 5 min and washed twice in PBS. Finally, the cells were stained with 7-AAD staining solution (Invitrogen) and analyzed by flow cytometry (collecting 25,000 events per sample).
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