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Researchers who sort objects that are unusually large (plankton, blast cells, early embryos), tiny (microparticles, bacteria, biological condensates) or highly asymmetric (sperm, myotubes), should first examine the intended instrument to ensure that it has been successfully used for that application in the past.
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A recommendation would be to make sure the instrument produces an output file that is compatible with the post-collection analysis software you intend to use. The software is often not as robust as the software from larger companies, particularly when monitoring day-to-day performance or comparison of data from multiple instruments working on a large-scale project (e.g., a clinical trial). They tend to take advantage of the development of smaller diode lasers and improvements in microfluidics. LSR-Fortessa, C6, Celesta ACEA BiosciencesĬompanies that specialize in the analysis of small particles (JSAN, Partec, Accuri) target the individual lab or else the lab that needs to do regular quality analysis on their cell sorters. Microalgae /plankton populations and communitiesĬirculating endothelial cells and precursorsīD® / FACSymphony / FACS Calibur / Aria / Canto The coverage of these topics, not exhaustive by any means, should allow the reader to understand the considerations that must be taken before embarking on a flow cytometric analysis of any of the applications listed in Table 1. This article focuses on 1) hardware choices and considerations, 2) practical aspects of flow cytometry and cell sorting, and 3) data analysis and software.
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Formal guidelines have also been published for specific applications. In addition, another article covers the major antibody suppliers that provide specific reagents for flow cytometric applications. There is a fine review of the theory behind flow cytometry that introduces the language of flow cytometry. Nitta N et altr described an optical-microfluidic-electrical-computational-mechanical system, named intelligent image-activated cell sorter (IACS), which integrated a three-dimensional on-chip hydrodynamic cell focuser, a frequency-division-multiplexed (FDM) microscope, a real-time intelligent image processor, and an on-chip dual-membrane push-pull cell sorter to achieve versatile and high-throughput single-cell acquisition. A recent publication describes an improved technique, "ghost cytometry," which may provide low-cost, fast, and accurate cell sorting. For example, Hafner AS et al used PASS, fluorescence-activated synaptosome sorting, to study the local protein synthesis in neuronal pre- and postsynaptic compartments. Cell sorting has been extended to sort cell organelles and fractions.
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Lastly, data analysis is tricky, particularly when dealing with low abundance targets. Specifically for cell sorters, there are problems of cell survival after pressure changes during droplet formation and collection, dilution of the sorted cells before reanalysis or culture, and the time delay it takes to obtain a sufficient number of viable cells. Technical issues persist around the detection of low abundance molecules in intracellular compartments, the lack of "universal" cell permeabilizing chemistries, confounding effects from cell autofluorescence, the overlap of emission spectra between fluorochromes, and unavailability of reagents for targeting molecules of interest. However, there remain numerous impediments, other than cost, to the further acceptance of the technology by many laboratories. Since the initial commercialization of Flow Cytometry (FC) and Fluorescence Activated Cell Sorting (FACS) in 1968, they have undergone significant improvements.