Guidelines for establishing a cytometry laboratory.

The purpose of this document is to provide guidance for establishing and maintaining growth and development of flow cytometry shared resource laboratories. While the best practices offered in this manuscript are not intended to be universal or exhaustive, they do outline key goals that should be prioritized to achieve operational excellence and meet the needs of the scientific community. Additionally, this document provides information on available technologies and software relevant to shared resource laboratories. This manuscript builds on the work of Barsky et al. 2016 published in Cytometry Part A and incorporates recent advancements in cytometric technology. A flow cytometer is a specialized piece of technology that require special care and consideration in its housing and operations. As with any scientific equipment, a thorough evaluation of the location, space requirements, auxiliary resources, and support is crucial for successful operation. This comprehensive resource has been written by past and present members of the International Society for Advancement of Cytometry (ISAC) Shared Resource Laboratory (SRL) Emerging Leaders Program https://isac-net.org/general/custom.asp?page=SRL-Emerging-Leaders with extensive expertise in managing flow cytometry SRLs from around the world in different settings including academia and industry. It is intended to assist in establishing a new flow cytometry SRL, re-purposing an existing space into such a facility, or adding a flow cytometer to an individual lab in academia or industry. This resource reviews the available cytometry technologies, the operational requirements, and best practices in SRL staffing and management.

Procedures for Flow Cytometry-Based Sorting of Unfixed Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infected Cells and Other Infectious Agents.

In response to the recent COVID-19 pandemic, many laboratories are involved in research supporting SARS-CoV-2 vaccine development and clinical trials. Flow cytometry laboratories will be responsible for a large part of this effort by sorting unfixed antigen-specific lymphocytes. Therefore, it is critical and timely that we have an understanding of risk assessment and established procedures of infectious cell sorting. Here we present procedures covering the biosafety aspects of sorting unfixed SARS-CoV-2-infected cells and other infectious agents of similar risk level. These procedures follow the ISAC Biosafety Committee guidelines and were recently approved by the National Institutes of Health Institutional Biosafety Committee for sorting SARS-CoV-2-infected cells. © 2020 International Society for Advancement of Cytometry.

Novel Impactor and Microsphere-Based Assay Used to Measure Containment of Aerosols Generated in a Flow Cytometer Cell Sorter.

Today's state-of-the-art cell sorting flow cytometers are equipped with aerosol containment systems designed to evacuate aerosols from the sort chamber during a sort. This biosafety device is especially important when the sort operator is sorting infectious or potentially infections samples. Hence, it is critical to evaluate the performance for this system in normal operation and in "failure" mode to determine the efficacy of containment. In the past decade, the most popular published method for evaluating containment has been the Glo-Germ bead procedure. These highly fluorescent and multisize particles can easily be detected on a microscope slide and enumerated using a fluorescent microscope. Collecting particles on this slide is accomplished using an Aerotech impactor. This sampler collects potentially escaping aerosols from the sort chamber before enumerating any particles. Although the Glo-Germ procedure has been adopted by many labs, there are several drawbacks with the procedure that have limited its adoption by cell sorter laboratories: The Aerotech impactor is a reusable device that requires rigorous cleaning between measurements. The surface area of the collection slide is large and difficult to scan on a fluorescence microscope. These beads produce a wide variation in sizes resulting in inconsistency in flow rates. Here, we describe a novel and replacement method utilizing a Cyclex-d impactor and Dragon Green beads. This method was compared for sensitivity of detection of escaped aerosols with a published method for aerosol detection which utilizes a UV-APS aerodynamic particle sizer and a UV-excitable dye. One of the advantages of the Cyclex-d system is the narrow-defined field of collection as compared to the standard Glo-Germ bead procedure, this means a smaller sampling area is used in the Cyclex-d impactor as compared to the AeroTech impactor. In addition, the sensitivity of detection was found to be better using the Cyclex-d collection device as compared to the standard Glo-Germ bead procedure. © 2018 International Society for Advancement of Cytometry.

Basin-Scale Observations of Monoterpenes in the Arctic and Atlantic Oceans.

We report novel in situ speciated observations of monoterpenes (α- and ß-pinene, myrcene, δ3-carene, ocimene, limonene) in seawater and air during three cruises in the Arctic and Atlantic Oceans, in/over generally oligotrophic waters. Oceanic concentrations of the individual monoterpenes ranged from below the detection limit of <1 pmol L-1 to 5 pmol L-1, with average concentrations of between 0.5 and 2.9 pmol L-1. After careful filtering for contamination, atmospheric mixing ratios varied from below the detection limit (<1 pptv) to 5 pptv, with averages of 0.05-5 pptv; these levels are up to 2 orders of magnitude lower than those reported previously. This could be at least partly due to sampling over waters with much lower biological activity than in previous studies. Unlike in previous studies, no clear relationships of the monoterpenes with biological variables were found. Based on our measured seawater concentrations and a global model simulation, we estimate total global marine monoterpene emissions of 0.16 Tg C yr-1, similar to a previous bottom-up estimate based on laboratory monoculture studies but 2 orders of magnitude lower than a previous top-down estimate of 29.5 Tg C yr-1.

Archaeal and bacterial communities respond differently to environmental gradients in anoxic sediments of a California hypersaline lake, the Salton Sea.

Sulfidic, anoxic sediments of the moderately hypersaline Salton Sea contain gradients in salinity and carbon that potentially structure the sedimentary microbial community. We investigated the abundance, community structure, and diversity of Bacteria and Archaea along these gradients to further distinguish the ecologies of these domains outside their established physiological range. Quantitative PCR was used to enumerate 16S rRNA gene abundances of Bacteria, Archaea, and Crenarchaeota. Community structure and diversity were evaluated by terminal restriction fragment length polymorphism (T-RFLP), quantitative analysis of gene (16S rRNA) frequencies of dominant microorganisms, and cloning and sequencing of 16S rRNA. Archaea were numerically dominant at all depths and exhibited a lesser response to environmental gradients than that of Bacteria. The relative abundance of Crenarchaeota was low (0.4 to 22%) at all depths but increased with decreased carbon content and increased salinity. Salinity structured the bacterial community but exerted no significant control on archaeal community structure, which was weakly correlated with total carbon. Partial sequencing of archaeal 16S rRNA genes retrieved from three sediment depths revealed diverse communities of Euryarchaeota and Crenarchaeota, many of which were affiliated with groups previously described from marine sediments. The abundance of these groups across all depths suggests that many putative marine archaeal groups can tolerate elevated salinity (5.0 to 11.8% [wt/vol]) and persist under the anaerobic conditions present in Salton Sea sediments. The differential response of archaeal and bacterial communities to salinity and carbon patterns is consistent with the hypothesis that adaptations to energy stress and availability distinguish the ecologies of these domains.