The HEp-2 and TU212 cells transiently transfected with miR-negative control (miR-NC), miR-196a mimics, miR-196a inhibitors, pEGFP-NC, and stably transfected with pEGFP-p27kip1 were harvested 48 h after transfection using trypsinization. Apoptotic cells were detected using an Annexin V/APC and propidium iodide (PI) apoptosis detection kit I (BD Pharmingen, San Diego, CA, USA) in accordance with the manufacturer’s recommendations. The cells were analyzed with a flow cytometry (FACScan; BD Biosciences) equipped with CellQuest software. Cells were distinguished into viable, dead, early apoptotic and apoptotic, and then the relative ratio of early apoptotic cells were compared with the control transfection cells from each experiment. Cells for cell-cycle analysis were stained with propidium iodide for 1 h and analyzed by FACS can flow cytometer and CellQuest 3.3 software. The percentage of the cells in G0/G1, S and G2/M phase were counted and compared [24 (link)].
Annexin 5 apc and propidium iodide apoptosis detection kit 1
Manufactured by BD
Sourced in United States
The Annexin V/APC and propidium iodide (PI) apoptosis detection kit is a laboratory tool used to detect and analyze apoptosis, a form of programmed cell death. The kit utilizes fluorescently-labeled Annexin V, which binds to phosphatidylserine exposed on the surface of apoptotic cells, and propidium iodide, a dye that stains the DNA of cells with compromised cell membranes. This combination allows for the identification and quantification of cells in early and late stages of apoptosis.
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2 protocols using annexin 5 apc and propidium iodide apoptosis detection kit 1
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Apoptosis and Cell Cycle Analysis
2
Annexin V-FITC Apoptosis Assay
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At 24-h post-transfection, apoptotic cells were detected using Annexin V/APC and propidium iodide (PI) apoptosis detection kit I (BD Pharmingen; BD Biosciences), according to the manufacturer's protocol. Briefly, NSCLC cells were collected after centrifugation at 400 × g for 10 min at room temperature, and then washed with cold PBS. Then, 1×105 cells/ml were stained with binding buffer, containing 5 µl Annexin V-FITC and 10 µl PI at 4°C in the dark for 15 min. Following the incubation, cell apoptosis was analyzed using a FACScan flow cytometer (BD Biosciences) and CellQuest software version 3.3 (BD Biosciences).
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