The Detailed Comparison of Cell Death Detected by Annexin V-PI Counterstain Using Fluorescence Microscope, Flow Cytometry and Automated Cell Counter in Mammalian and Microalgae Cells.

The evaluation of cell wellness is an important task for molecular biology research. This mainly comprises the assessment for morphology and viability of culturing cells. Annexin V-Propidium iodide counterstaining has been currently one of the common and easy methods to discriminate apoptotic and necrotic cell profiles. The method is operated by fluorescence-based detection of counterstain via laser beam-employed instruments including flow cytometer, fluorescence microscope and automated cell counter. The detection is primarily conducted based on the same principle; however the efficiency of instruments may vary. Here we evaluated the efficiency of those instruments for the clear-cut detection of cell death through various mammalian and microalgae cell lines. To the best of our knowledge, this is the first study revealing comparative analyses of apoptotic and necrotic cells in mammalian and microalgae cells using Annexin V-PI counterstain detected by flow cytometer, fluorescence microscope and automated cell counter. Fluorescence microscope and cell counter instruments were also tested and compared for the traditional trypan blue-based cell viability detection performance. For these, cell death was induced by UV-irradiation and/or bee venom for mammalian (pancreatic cancer, metastatic breast cancer and mouse fibroblasts) and microalgae cells (Chlorella vulgaris), respectfully. Findings postulated that automated cell counter and fluorescence microscopy revealed similar patterns for the detection by both counterstain and trypan blue in mammalian cells. Interestingly, flow cytometry did provide an accurate and significant detection for only one mammalian cell line when UV-treatment was followed by routine Annexin V-Propidium iodide counterstaining. Unlike, only flow cytometry revealed a significant change in the detection of death of microalgae cells by Annexin V-Propidium iodide method, but both Annexin and conventional trypan blue methods were not applicable for the automated cell counter and microscopic detections for microalgae cells. The related outputs propose that the obtaining reliable quantitation strongly depends on cell type and instruments used. These suggest the necessity of optimization and validation endeavors before any cell death detection initiative. The analytical outcomes present insights into detailed assessment of cell death detection of eukaryotic cells and provide a direction to researchers to consider.