Abstract
BackgroundFor in vitro culture of plant and animal cells, one of the critical steps is to adjust the initial cell density. A typical example of this is isolated microspore culture, where specific cell densities have been determined for different species. Out of these ranges, microspore growth is not induced, or is severely reduced. A similar situation occurs in many other plant and animal cell culture systems. Traditionally, researchers have used counting chambers (hemacytometers) to calculate cell densities, but little is still known about their technical advantages. In addition, much less information is available about other, alternative methods. In this work, using isolated eggplant microspore cultures and fluorescent beads (fluorospheres) as experimental systems, we performed a comprehensive comparison of six methods to calculate cell densities: (1) a Neubauer improved hemacytometer, (2) an automated cell counter, (3) a manual-counting method, and three flow cytometry methods based on (4) autofluorescence, (5) propidium iodide staining, and (6) side scattered light (SSC).ResultsOur results show that from a technical perspective, hemacytometers are the most reasonable option for cell counting, which may explain their widely spread use. Automated cell counters represent a good compromise between precision and affordability, although with limited accuracy. Finally, the methods based on flow cytometry were, by far, the best in terms of reproducibility and agreement between them, but they showed deficient accuracy and precision.ConclusionsTogether, our results show a thorough technical evaluation of each counting method, provide unambiguous arguments to decide which one is the most convenient for the particular case of each laboratory, and in general, shed light into the best way to determine cell densities for in vitro cell cultures. They may have an impact in such a practice not only in the context of microspore culture, but also in any other plant cell culture procedure, or in any process involving particle counting.
Highlights
For in vitro culture of plant and animal cells, one of the critical steps is to adjust the initial cell density
For evaluation of precision, accuracy, reproducibility and concordance between methods, a total of 17 microspore cultures were performed and measured using the different methods tested in this work
Microspore density was estimated for each culture at days 3 and 18. 3 day-old cultures are formed by microspores induced towards embryogenesis, together with non-induced, dead and arrested individuals
Summary
For in vitro culture of plant and animal cells, one of the critical steps is to adjust the initial cell density. A typical example of this is isolated microspore culture, where specific cell densities have been determined for different species. In this work, using isolated eggplant microspore cultures and fluorescent beads (fluorospheres) as experimental systems, we performed a comprehensive comparison of six methods to calculate cell densities: (1) a Neubauer improved hemacytometer, (2) an automated cell counter, (3) a manual-counting method, and three flow cytometry methods based on (4) autofluorescence, (5) propidium iodide staining, and (6) side scattered light (SSC). As far as we know, flow cytometry has only been used to identify populations of embryogenic and non-embryogenic microspores [25] and to identify cellulose deposition in embryogenic microspores [26] These studies did not use this method for the initial adjustment of microspore density. Other methods based on automated cell counting have not been described in the literature
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