After treatment, cells collected were mixed with 5 μL Annexin V-APC and 5 μL 7-AAD according to the protocol of the APC Annexin V Apoptosis Detection Kit I (BD Pharmingen). Finally, the abovementioned mixture was added into 400 μL of 1× binding buffer and measured by flow cytometry in 1 h. These data were analyzed by WinMDI 2.9 software.
Annexin 5 apc apoptosis detection kit 1
Manufactured by BD
Sourced in United States
The Annexin V-APC Apoptosis Detection Kit is a laboratory reagent used to detect and quantify apoptosis, a type of programmed cell death. The kit contains Annexin V, a protein that binds to phosphatidylserine, a molecule that is exposed on the surface of cells undergoing apoptosis. The Annexin V is conjugated to the fluorescent dye APC, allowing for the detection and measurement of apoptotic cells using flow cytometry or fluorescence microscopy.
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7 protocols using annexin 5 apc apoptosis detection kit 1
1
Annexin V-APC and 7-AAD Apoptosis Assay
2
Measuring Cell Survival and Apoptosis
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For survival assays, cells were seeded at the density of 2 × 105 cells per well in six-well plates in culture medium (2 ml) containing fetal bovine serum (FBS). After 24 h, cells were washed twice with PBS and then incubated in serum-free medium. After 8 days, the cells were collected and the viable cells were counted after Trypan blue staining.
For Annexin V apoptosis assays, the cells were grown in low serum (1%) or serum-free medium for 5 days. The cells were separately washed twice with cold PBS and stained with binding buffer containing fluorescein-labelled Annexin V and with propidium iodide (PI; APC Annexin V Apoptosis Detection Kit I, BD Pharmingen) following manufacturer’s instructions. The cells that were positive for apoptosis (APC annexin V positive, PI negative or APC annexin V positive, PI positive) were analysed by flow cytometry.
For Annexin V apoptosis assays, the cells were grown in low serum (1%) or serum-free medium for 5 days. The cells were separately washed twice with cold PBS and stained with binding buffer containing fluorescein-labelled Annexin V and with propidium iodide (PI; APC Annexin V Apoptosis Detection Kit I, BD Pharmingen) following manufacturer’s instructions. The cells that were positive for apoptosis (APC annexin V positive, PI negative or APC annexin V positive, PI positive) were analysed by flow cytometry.
3
Comprehensive Immunophenotyping of Murine Cells
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Mouse cells staining was performed using fluorochrome-labeled antibodies specific to MHC class II, CD11b, F4/80, Gr-1, CD40, CD45, CD80, and CD86 (Thermo Fisher Scientific, Irwindale, CA). Unspecific binding was blocked with anti-FcγIII/IIR (Thermo Fisher Scientific). For viability assessment, cells were stained using APC Annexin V apoptosis detection kit I (BD Biosciences, Franklin Lakes, NJ). For EVs phenotyping, isolated vesicles were incubated with anti-CD63 beads (Thermo Fisher Scientific) according to manufacturer’s instruction and then stained with fluorochrome-labeled CD9, CD63, and CD81 antibodies (Thermo Fisher Scientific). Fluorescence data were acquired on Attune NxT Flow Cytometer (Thermo-Fisher) and analyzed using FlowJo software (TreeStar).
4
Apoptosis Analysis by Annexin V-APC and PI
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Annexin V and propidium iodide (PI) staining was carried out using Annexin V-APC apoptosis detection kit I (BD pharmingenTM, San Diego, CA, USA). HSC-T6 and LX-2 cells (2×105 cells/mL) were exposed to selonsertib (10-50 μM) for 48 h, and then cellular apoptosis was detected by fluorescent staining. Prepared cells were incubated in Annexin V-APC and PI solution for 15 min in the dark, and the stained cells were instantly analyzed using a FACS verse flow cytometer (BD Biosciences, San Jose, CA, USA).
5
Cell Cycle and Apoptosis Analysis
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SGC-7901/KD, NCI-N87/KD cells and control cells were collected and cell cycle and apoptosis was assessed by flow cytometry. For cell cycle analysis, cells were fixed with 75% ethanol and stored at 4°C overnight. The following day, fixed cells were washed with PBS, treated with RNase A (50 µg/ml) and stained with propidium iodide (PI) (50 µg/ml) for 30 min in the dark. The stained cells were analyzed by flow cytometry (FACSCalibur, Becton-Dickinson).
For apoptosis analysis, an Annexin V-APC Apoptosis Detection Kit I (BD Pharmingen, USA) was used according to the manufacturer's instructions. PI and Annexin V-APC double staining was performed and cells were analyzed by flow cytometry (FACSCalibur, Becton-Dickinson), using the Cell Quest software (Becton-Dickinson). Annexin V-APC-positive and PI-negative cells were defined as undergoing apoptosis. All experiments were performed in triplicate and the average values of these groups were calculated.
For apoptosis analysis, an Annexin V-APC Apoptosis Detection Kit I (BD Pharmingen, USA) was used according to the manufacturer's instructions. PI and Annexin V-APC double staining was performed and cells were analyzed by flow cytometry (FACSCalibur, Becton-Dickinson), using the Cell Quest software (Becton-Dickinson). Annexin V-APC-positive and PI-negative cells were defined as undergoing apoptosis. All experiments were performed in triplicate and the average values of these groups were calculated.
6
Apoptosis and Necrosis Assay for PBMCs
7
Cell Death and Senescence Assays
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