Viable cell numbers were counted based the Trypan blue exclusion (Corning Inc., Corning, NY, USA). Cell apoptosis was detected by staining with Annexin V-Fluorescein isothiocyanate (FITC) and 7-AAD (BioLegend). Briefly, 2 × 105 primary mSMG cells were incubated with recombinant murine IL-22 (20 ng/mL) for 3 days. The cells were subsequently stained with Trypan blue solution for viable cell enumeration, or incubated with Annexin V-FITC plus 7-AAD (BioLegend) for apoptosis detection. The cells were then analyzed using a BD FACSAria III cell sorter/flow cytometer and the Flowjo software to determine the percentage of cells stained positive for Annexin V and/or 7-AAD.
Annexin 5 fitc plus 7 aad
Manufactured by BioLegend
Annexin V-FITC plus 7-AAD is a laboratory reagent used for the detection and analysis of apoptosis in cells. Annexin V is a calcium-dependent phospholipid-binding protein that has a high affinity for phosphatidylserine, which is exposed on the surface of apoptotic cells. FITC (fluorescein isothiocyanate) is a fluorescent dye used to label the Annexin V. 7-AAD (7-Aminoactinomycin D) is a DNA-binding dye that can be used to identify necrotic cells. This combination of reagents allows for the identification and quantification of apoptotic and necrotic cells in a sample.
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2 protocols using annexin 5 fitc plus 7 aad
1
Apoptosis Quantification of Primary mSMG Cells
2
Flow cytometric analysis of cell death
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MS-275 or IFN-λ-treated U87 cells in six-well plates were trypsinized and stained with propidium iodide (Fisher) or Annexin V-FITC plus 7-AAD (BioLegend). Stained cells were washed with PBS, and fluorescence was measured with a 13-color Stratedigm S1000Ex flow cytometry platform. Laser light scatter was used to gate out dead or dying cells. For HSV-1 and VSV experiments, infected astrocytes were fixed with PFA, stained for live/dead cells (BioLegend), and examined using the Stratedigm flow cytometer for GFP expression. Data were analyzed with FlowJo Software v8.8.7 (TreeStar).
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