My paper, "Preparation of Cuvette-Based Sorters for Sorting Submicron Microbial Cells and Viruses from Environmental and Biological Samples," has been published in Current Protocols in the Cytometry section!
This paper shows you how to prepare a cuvette-based sorter for submicron or small particle fluorescence-activated cell sorting (FACS). To my knowledge, it is the most complete and detailed resource published to date that holistically documents instrument preparation and cleaning for this specific purpose. In addition to providing methods, it also formally documents information that largely has been shared informally among researchers at conferences, in the lab, and in community forums for the field of cytometry, but is essential for performing the experiments and reproducing results. This paper serves as a foundational resource for future efforts to develop best practices for instrument cleaning and preparation in various contexts, but especially for submicron or small particle sorting for downstream experimentation that requires genomic sequencing (such as microbiome analysis, characterization of uncultivated microbes, and much more).
When I had the initial idea to publish these protocols and discussed it with my co-authors, the goal was to create a resource that fills a gap in the literature, a gap that I noticed as soon as these challenging experiments came my way as a staff scientist in a Shared Resource Laboratory (SRL) setting. Complete methods and step-by-step instructions for submicron or small particle sorting were not available; methods sections were incomplete, were for mismatched instrumentation that was no longer being serviced by the manufacturer and not what I had in my lab, and/or were scattered throughout the literature in bits and pieces. Much of the literature around submicron or small particle work is geared toward analysis-only at this time. As I attended conferences outside of the world of flow cytometry SRLs, and spoke with more colleagues, I heard numerous stories about researchers with these types of samples being turned away from SRLs.
I started with the baseline of my own existing protocols for routine microbial and yeast sorting, and then did the work needed to adapt them to similarly challenging submicron or nanoscale samples on a BD FACS Aria Fusion. Then, I documented them in detail over years to prepare them for publication and share them widely with the goal of empowering researchers, improving reproducibility in the long-term, and encouraging other groups to do the same.
This paper and the protocols contained within it are suitable for sorting diverse submicron, small particle, nanoscale samples including, but not limited to: virus-like particles, ultramicrobacteria, extremophiles, extracellular vesicles, exosomes, minicells, microplastics or nanoplastics including nanoparticle beads, and more. These protocols will also work for instrumentation like the BD FACS Symphony S6 and the BD FACS Discover S8 that has the same fluidic path as the BD FACS Aria II, III, and Fusion. They can be adapted for other instrumentation in the future.
Validation of sorting is discussed as well; submicron, small particle, nanoscale sorting typically benefits from and requires validation with other methods including (but not limited to) microscopy, cultivation, or genomic sequencing
These protocols are also very useful for general purpose sorting as well as day-to-day maintenance and decontamination of cell sorters. Much of the information about cleaning agents is relevant to daily use and other applications with mammalian cells and more traditional samples.
Here is the citation for the paper:
Tijerina, J.C., Martinez-Hernandez, F., Beilinson, V., Finney, O., Orphan, V.J., & Diamond, R. A. (2025). Preparation of cuvette-based sorters for sorting submicron microbial cells and viruses from environmental and biological samples. Current Protocols, 5, e70176. doi: 10.1002/cpz1.70176
Full-text pdf is available:
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