Apoptosis and necrosis play an important role in various aspects of preclinical pharmaceutical drug discovery and validation. The ability to quickly determine the cytotoxic effect of chemical compounds on cancer cells allows researchers to efficiently identify potential drug candidates for further development in the pharmaceutical discovery pipeline. Recently, a new imaging cytometry system has been developed by Nexcelom Bioscience LLC (Lawrence, MA, USA) for fluorescence-based cell population analysis. Currently, fluorescence-based cell death assays have not been demonstrated by the Cellometer system, which can potentially provide a quick, simple, and inexpensive alternative method for smaller biomedical research laboratories. In this study, we demonstrate for the first time the use of Cellometer imaging cytometry for necrosis/apoptosis detection by studying the dose-response effect of heat and drug-induced cell death in Jurkat cells labeled with annexin V-FITC (apoptotic) and propidium iodide (necrotic). The experimental results were evaluated to validate the imaging cytometric capabilities of the Cellometer system as compared to the conventional flow cytometry. Similar cell population results were obtained from the two methods. The ability of Cellometer to rapidly and cost-effectively perform fluorescent cell-based assays has the potential of improving research efficiency, especially where a flow or laser scanning cytometer is not available or in situations where a rapid analysis of data is desired.
Monitoring populations of immune cell subsets, such as B lymphocytes, T lymphocytes, NK cells, NKT cells, Treg cells, monocytes and dendritic cells, is important in many different infectious diseases, including HIV/AIDS, to assess response and recovery to treatment and therapy. In the last decade, multicolor flow cytometry has greatly increased the capabilities and consistency of immunophenotyping and immunomonitoring. However, limitations still preclude a wider adaption of >10 color flow cytometry, largely due to the limited availability of fluorophores with flexible utility in a complex panel. Although instruments with 18-20 parameters are now increasingly common, until recently, the available reagents had significant practical limitations, such as limited brightness and permeability, and undesirable cross-beam excitation, resulting in reduced sensitivity when detecting lowly abundant antigens in these assaulted channels. The Brilliant Violet™ family of fluorescent probes, released in 2011, overcomes these limitations, with significantly increased overall brightness and signal-to-noise. Here, we present a 15 color/ 17 parameter flow cytometry assay, introducing Brilliant Violet™ 605 and Brilliant Violet 650™ in our expanding family of fluorescent polymers. This family of fluorescent probes will be instrumental in making a wide array of commercially available conjugates with a broad range of brightness and no limitations for intracellular detection.
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