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Improved strategy for jet-in-air cell sorting with high purity, yield, viability, and genome stability.

ABSTRACT: Flow cytometric sorting is a vital tool in biological research and clinical diagnostics. Theoretically, a high-speed jet-in-air sorter is a fluorescent-activated cell sorting sorter that ideally processes cells with high purity, yield, and viability. However, high-speed jet-in-air sorting is a complex process due to its inherent requirements for high fluidic stability and electronic and timing precision. Here, we report that an additional manual correction of drop delay leads to improved cell yield. Adding 2% FBS to the loading buffer had no significant effect on the fate of sorted cells in 4 h. However, the addition of a suitable concentration of FBS/BSA in the collecting buffer resulted in a notable increase in cell count and proliferation and a significant decrease in cell apoptosis for cell lines and primary cells. Moreover, the level of gene expression remained steady in the 5% FBS collecting buffer. In summary, here we demonstrate techniques that can be easily followed to refine sorted yields of healthy cells.

SUBMITTER: Song X

PROVIDER: S-EPMC8409286 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe integration of first- and second-generation bioethanol processes has the potential to accelerate the establishment of second-generation bioethanol on the market. Cofermenting pretreated wheat straw with a glucose-rich process stream, such as wheat grain hydrolysate, in a simultaneous saccharification and fermentation process could address the technical issues faced during the biological conversion of lignocellulose to ethanol. For example, doing so can increase the final ethanol concentration in the broth and mitigate the effects of inhibitors formed during the pretreatment. Previous research has indicated that blends of first- and second-generation substrates during simultaneous saccharification and fermentation have synergistic effects on the final ethanol yield, an important parameter in the process economy. In this study, enzymatic hydrolysis and simultaneous saccharification and fermentation were examined using blends of pretreated wheat straw and saccharified wheat grain at various ratios. The aim of this study was to determine the underlying mechanisms of the synergy of blending with regard to the yield and volumetric productivity of ethanol.ResultsReplacing 25% of the pretreated wheat straw with wheat grain hydrolysate during simultaneous saccharification and fermentation was sufficient to decrease the residence time needed to deplete soluble glucose from 96 to 24 h and shift the rate-limiting step from ethanol production to the rate of enzymatic hydrolysis. Further, a synergistic effect on ethanol yield was observed with blended substrates, coinciding with lower glycerol production. Also, blending substrates had no effect on the yield of enzymatic hydrolysis.ConclusionsThe effects of substrate blending on the volumetric productivity of ethanol were attributed to changes in the relative rates of cell growth and cell death due to alterations in the concentrations of substrate and pretreatment-derived inhibitors. The synergistic effect of substrate blending on ethanol yield was attributed in part to the decreased production of cell mass and glycerol. Thus, it is preferable to perform simultaneous saccharification and fermentation with substrate blends rather than pure substrates with regard to yield, productivity, and the robustness of the process.

Project description:ObjectivesIn COVID-19, transfer of respiratory materials transmits disease and drives the pandemic but the interplay of droplet and aerosol physics, physiology and environment is not fully understood. To advance understanding of disease transmission mechanisms and to find novel exposure minimisation strategies, we studied cough-driven material transport modes and the efficacy of control strategies.DesignComputer simulations and real-world experiments were used for integrating an intensive care setting, multiphysics and physiology. Patient-focused airflow management and air purification strategies were examined computationally and validated by submicron particle exhalation imaging in volunteers.SettingHospital setting during a respiratory virus pandemic with transmission by respiratory droplets and aerosols.ParticipantsHealthy volunteers.Outcome measuresDistribution of, and exposure to, potentially infectious respiratory secretions.ResultsRespiratory materials ejected by cough exhibited four transport modes: long-distance ballistic, short-distance ballistic, 'jet rider' and aerosol modes. Interaction with air conditioning driven flow contaminated a hospital room rapidly. Different than large droplets or aerosols, jet rider droplets travelled with the turbulent air jet initially, but fell out at a distance, were not well eliminated by air conditioning and exposed bystanders at larger distance and longer time; their size predisposes them to preferential capture in the nasal mucosa, the primordial COVID-19 infection site. 'Cough shields' captured large droplets but induced lateral dispersion of aerosols and jet riders. An air purification device alone had limited efficacy. A 'Shield and Sink' approach combining cough shields with 'virus sinks' minimised exposure to all secretions in modelling and real-life experiments.ConclusionsJet riders have characteristics of highly efficient respiratory infection vectors and may play a role in COVID-19 transmission. Exposure to all droplet types can be minimised through an easily implemented Shield and Sink strategy.

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Improved strategy for jet-in-air cell sorting with high purity, yield, viability, and genome stability.

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