Thymocytes were freshly isolated from WT, RORγt−/−, or RORγtK31R/K31R mice and cultured in RPMI 1640 medium supplemented with 10% FBS, 100 U/ml penicillin–streptomycin, and 2 mM L-glutamine at 1 × 106 cells/ml. Thymocytes were incubated at 37 °C with 5% CO2. Dead cells were detected using Annexin V-PE and 7-AAD staining (BD Bioscience).
Annexin 5 pe and 7 aad staining
Manufactured by BD
Annexin V-PE and 7-AAD staining is a laboratory technique used to detect and quantify cell apoptosis, or programmed cell death. Annexin V is a protein that binds to phosphatidylserine, a lipid that is exposed on the surface of cells undergoing apoptosis. 7-AAD is a dye that can only enter cells with compromised cell membranes, such as late-stage apoptotic or necrotic cells. By using both Annexin V-PE and 7-AAD, researchers can distinguish between early apoptotic, late apoptotic, and necrotic cells in a sample.
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5 protocols using annexin 5 pe and 7 aad staining
1
Isolation and Culture of Thymocytes
2
Thymocyte Apoptosis Assay in Mice
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Thymocytes were freshly isolated from wild-type, RORγt−/−, or RORγtM/M mice and cultured in RPMI 1640 medium supplemented with 10% FBS, 100 U/ml penicillin-streptomycin, 2 mM L-glutamine at 1×106 cells/ml. Thymocytes were incubated at 37°C with 5% CO2. Dead cells were detected by Annexin V-PE and 7-AAD staining (BD Bioscience), as described previously 34 (link).
3
Cell Cycle and Apoptosis Analysis
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The percentages of cell cycle distribution were evaluated by PI staining. The cells were washed with precooled PBS and fixed overnight with 70% ethanol at 4°C. After rewashing with precooled PBS, 5% propidium iodide (Beyotime, China) was used for labelling the cells and incubated at 37℃ in dark for 30 min. The cell cycle was then analyzed by flow cytometer. Apotosis detected using an Annexin V fluorescein isothiocyanate kit according to the protocol. The cells were resuscitated with 100 μL 1× binding buffer, and the cell concentration was kept at about 1 × 106 cells /mL. Annexin V-PE and 7-AAD staining (BD) were added to the 1× binding buffer to prepare the cell staining solution (Annexin V-PE: 7-AAD: 1× binding buffer = 1:1:20). The cells were thereafter stained in the dark for 15 min, and analyzed by flow cytometry within 1 h. All samples were analyzed employing FACSCalibur (BD) with appropriate software (ModFit LT; BD, Topsham, ME) and the FlowJo (Ashland, OR) software were used for data analysis.
4
Thymocyte Culture and Viability Analysis
5
Thymocyte Apoptosis Assay in Mice
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