The Annexin V binding assay was performed according to manufacturer’s instruction using Annexin V-FITC or Annexin V-PE detection kit I (BD Biosciences, San Diego, CA, USA). Cells were harvested by trypsinization and washed twice with cold PBS. Cell pellets were re-suspended with 100 μL binding buffer at a density of 1×105 cells per ml and incubated with 5μL of FITC or PE-conjugated AnnexinV and 5 μL of propidium iodide (PI) or 7-AAD for 15 min at room temperature in the dark. 400 μl of 1X binding buffer was added to each sample tube, and immediately the samples were analyzed by BD FACSCanto II Flow Cytometer (BD Biosciences, USA).
Annexin 5 pe detection kit 1
Manufactured by BD
Sourced in United States
The Annexin V-PE detection kit is a laboratory tool used to identify and quantify apoptotic cells. It contains Annexin V, a protein that binds to phosphatidylserine, a molecule exposed on the surface of cells undergoing programmed cell death. The kit also includes Propidium Iodide, a dye that stains the nuclei of dead cells. The kit allows for the detection and analysis of apoptotic cells using flow cytometry or fluorescence microscopy.
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4 protocols using annexin 5 pe detection kit 1
1
Annexin V-Based Cell Apoptosis Assay
2
Medulloblastoma Cell Apoptosis Assay
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Medulloblastoma cells were stained with CFSE according to the supplier's instructions. Daoy (3×105/well), MEB-Med8A (5×105/well) D283 Med (5×105/well) and D341 Med (5×105/well) cells were seeded in 6-well cell culture dishes in complete medium. After overnight culture, the cells were treated with MKIs at concentrations corresponding to patient plasma levels for a 24h, 48h or 72h period. Thereafter floating and attached cells were collected and stained with 7-AAD and Annexin V-Antibody (Annexin V-PE Detection Kit I, BD Bioscience) and analysed by flow cytometry (Navious, Beckman Coulter). Proliferation was traced by CFSE staining and apoptosis was detected by combined 7AAD/Annexin V staining and calculated in percent of control.
3
Apoptosis and Cell Cycle Analysis of Lentiviral Transfection
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For the analysis of spontaneous apoptosis, the cells in the lentiviral transfection groups (SH1 and SH2), the untransfected group (Hut-78), and the scrambled shRNA sequence group (SH0) were seeded at 4×105/ml in 2 ml RPMI-1640 medium for 24 h, prior to the cells being washed with cold PBS and fixed in cold 70% ethanol overnight at −20°C. Next, the cells were suspended in 100 µl PBS, and then stained with 5 µl 7-aminoactinomycin D and 5 µl phycoerythrin-conjugated Annexin V using the Annexin V-PE Detection kit I (BD Pharmingen) at room temperature for 15 min, and quantified by flow cytometric analysis on a FACScan flow cytometer (BD Biosciences). Cell cycle analysis was performed using PI-mediated flow cytometry. A total of 5×105 transfected cells (SH1 and SH2), as well as control cells (Hut-78 and SH0) were collected, fixed and permeabilized with 100% ethanol for 1 h at 4°C. Following treatment with DNase-free RNase, the cells were stained with 50 µg/ml PI for 1 h at room temperature. Distribution of the cell-cycle phases was determined using a FACScan flow cytometer (BD Pharmingen). For each sample, 10,000 gated events were obtained. Flow cytometry data were analyzed using CellQuest Pro and ModFit v3.3 software (BD Biosciences). The experiments were performed in triplicate and repeated at least three times.
4
Medulloblastoma Cell Apoptosis Assay
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Medulloblastoma cells were stained with CFSE according to the supplier's instructions. Daoy (3 × 105/6 well), MEB-Med-8A (5 × 105/well), D283 Med (5 × 105/well) and D341 Med (5 × 105/well) cells were seeded in 6-well cell culture dishes in complete medium. After overnight culture, the cells were treated with different concentrations of GDC-0941 for a 24 and 48 h period. Thereafter floating and attached cells were collected and stained with 7-AAD and Annexin V antibodies (Annexin V-PE detection kit I, BD Bioscience) and analysed by flow cytometry (Navios, Beckman Coulter). Proliferation was traced by CFSE staining and apoptosis was detected by combined 7AAD/Annexin V staining and calculated in percent of control.
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