Cells were grown on a 60-mm tissue culture dish at a density of about 5.0 × 105 cells/well. They were treated for 48 h with the repurposed drug or its combination with 10-µM gefitinib. At the end of the treatment, both floating and attached cells were collected and washed twice with ice-cold phosphate buffer solution. The extent of apoptosis was determined by using the APC annexin-V apoptosis kit (BD Bioscience, San Jose, CA, USA) according to the manufacturer’s instructions. Cells positive for both annexin V and 7-AAD were considered apoptotic.
Annexin 5 apc apoptosis kit
Manufactured by BD
Sourced in United States
The Annexin V-APC Apoptosis Kit is a laboratory product designed to detect and quantify apoptosis, a programmed cell death process. The kit utilizes Annexin V, a protein that binds to phosphatidylserine, a molecule that is translocated to the outer cell membrane during apoptosis. Annexin V is conjugated with the fluorescent dye APC (allophycocyanin) to enable detection and measurement of apoptotic cells using flow cytometry or fluorescence microscopy.
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5 protocols using annexin 5 apc apoptosis kit
1
Quantifying Apoptosis in Drug-Treated Cells
2
Cell Cycle and Apoptosis Analysis
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RPCs were trypsinized and fixed by 70% ice-cold ethanol. RNase A (25 mg/ml) was used to treat cells for 30 min at 37 °C to eliminate RNA. Cells were stained with 50 mg/ml propidium iodide (PI) for 10 min at room temperature load to FACS Calibur flow cytometer (BD Biosciences, USA) to analysis of cell cycle. Data acquisition was performed using CellQuest software (BD Biosciences, USA), and the percentage of cells in the G0/G1, S, and G2/M phases was calculated using the Modfit. For apoptosis assay, cells were stained and analyzed with PI- and APC-conjugated Annexin V using an APC Annexin V Apoptosis kit (BD Biosciences, USA) according to the manufacturer’s protocol.
3
Nanoparticle-Induced Oxidative Stress Response
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The cells were washed with Dulbecco's phosphate-buffered saline (PBS, Bioshop, UK) and detached from the flask's surface using 0.25% trypsin with 0.02% EDTA (Sigma-Aldrich, Poland). Cells were cultured in a 24 well cell culture tray, and when the cells were 75–80% confluent (approximately 2 × 105 cells per well), they were treated with increasing concentrations of nanoparticles (0.05 mg mL−1, 0.1 mg mL−1 and 1.5 mg mL−1) for 24 and 48 hours. After the incubation with nanoparticles, the cells were exposed or not exposed to UV light (λ = 265 nm) for 1 hour to simulate an oxidative insult. Afterwards L929 cells were detached from the cultured well, washed with PBS and collected in 12 × 75 mm flow cytometric tubes. Subsequently, L929 fibroblast cells were stained with an Annexin V-APC Apoptosis Kit (BD Pharmingen™ Cat no 556547) and with propidium iodide (PI).
4
Annexin V-APC Apoptosis Assay
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Cell apoptosis was detected by using the annexin V–APC Apoptosis Kit (BD Biosciences). Briefly, cells were stained with annexin V–fluorescein isothiocyanate and 7-aminoactinomycin D (7-AAD) according to the manufacturer’s instruction. After incubation in the dark for 15 min at room temperature, stained cells were then analyzed by flow cytometer (BD Biosciences).
5
Annexin V-APC and PI Apoptosis Assay
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After PBS washing, cells were stained with Annexin V-APC Apoptosis Kit (BD Pharmingen™ Cat no 556,547) and propidium iodide. Stained cells, acquired by FACS Canto (Becton Dickinson, San Jose, USA) flow cytometry were analyzed by Diva Software 6.3. For each experimental point, 30.000 total events were acquired. The percentage of early, late apoptotic, and necrotic cells was analyzed using Microsoft Office Excel 2017 and GraphPad Prism 7.0 software. Apoptosis was also quantified with the “Cell Death detection ELISA kit” (Roche).
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