Cells were stimulated for 24 hours and following the incubation were washed with PBS by centrifugation at 400 ×g for 5 minutes. The supernatant was discarded and the cells were resuspended in 7-AAD solution (50 μg/mL, eBioscience) and incubated for 30 minutes at 4°C. Cells were kept on ice and immediately analysed by flow cytometry.
7 aad solution
Manufactured by Thermo Fisher Scientific
Sourced in Ireland
7-AAD solution is a fluorescent dye used for cell cycle analysis and cell viability assessment in flow cytometry. It binds to DNA and can be used to distinguish between live, apoptotic, and necrotic cells.
Automatically generated - may contain errors
4 protocols using 7 aad solution
1
Cell Viability Assay by Flow Cytometry
2
Quantifying Apoptotic Cell Populations
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Cells were stimulated for 24 h and, following the incubation, they were washed with PBS by centrifugation at 400× g for 5 min. The supernatant was discarded, and the cells were resuspended in 7-AAD solution (50 μg/mL, eBioscience, San Diego, CA, USA) and incubated for 20 min at 4 °C. Cells were kept on ice and immediately analyzed on an ADP Cyan flow cytometer (Beckman Coulter, Brea, CA, USA). The flow cytometry data were analyzed using FlowJo V10 (Tree Star, Ashland, OR, USA).
3
Cytometric Analysis of Necrotic Leukocytes
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20 µl blood samples were stained with 0.2 µg CD45-PE, clone 1.11.32, mouse IgG1 (Thermo Fisher Scientific, Gloucester, UK) for 30 min on ice in the dark. The red blood cells were lysed by adding 600 μl EasyLyse (1:20 with dH2O, DAKO, Alere, Cheshire, UK), followed by vortexing and 15 min incubation at room temperature in the dark. Necrotic cells were stained with 0.1 µg 7AAD solution (eBioscience, Hatfield, Ireland, UK) at room temperature in the dark for 15 min before 60 sec acquisition on the Navios flow cytometer (Beckman Coulter, High Wycombe, UK).
Leukocytes were gated and displayed on a contour density dot plot with 7AAD against CD45-PE. The healthy cell gate was set on the CD45 + population with high SSC. Any events to the right of this border were considered 7AAD-positive necrotic leukocytes.
Fragmented white blood cells (LMPs) still display CD45 but no longer contain DNA and because of their reduced size and complexity, and therefore lower side scatter, end up below the healthy leukocytes on an SSC axes in the gate designated MP (Supplementary Figure 1).
Leukocytes were gated and displayed on a contour density dot plot with 7AAD against CD45-PE. The healthy cell gate was set on the CD45 + population with high SSC. Any events to the right of this border were considered 7AAD-positive necrotic leukocytes.
Fragmented white blood cells (LMPs) still display CD45 but no longer contain DNA and because of their reduced size and complexity, and therefore lower side scatter, end up below the healthy leukocytes on an SSC axes in the gate designated MP (Supplementary Figure 1).
4
Characterizing Blood Cell Populations
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Blood samples from time points 5, 120, 240, and 360 min were stained with CD45-PE (Thermo Fisher Scientific, Gloucester, UK) for 30 min on ice in the dark. The red blood cells were lysed by adding EasyLyse (1:20 with dH 2 O, DAKO, Alere, Cheshire, UK), followed by vortexing and 15 min incubation at room temperature in the dark. Necrotic cells were stained with 7AAD solution (eBioscience, Hatfield, Ireland, UK) at room temperature in the dark for 15 min before acquisition. As a positive control for 7AAD staining of dead cells, 1 ml baseline blood was treated with Staurosporin solution (Sigma-Aldrich) at room temperature for at least 4 h prior to staining with CD45-PE and 7AAD. Untreated blood was singlestained with CD45-PE and was used to set the 7AAD-gate.
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