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Cooper M.S.,University of Washington | Hardin W.R.,University of Washington | Petersen T.W.,Advanced Cytometry Group | Cattolico R.A.,University of Washington
Journal of Bioscience and Bioengineering | Year: 2010

We report here that BODIPY 505/515, a green lipophilic fluorescent dye, serves as an excellent vital stain for the oil-containing lipid bodies of live algal cells. BODIPY 505/515 vital staining can be used in combination with fluorescent activated cell sorting to detect and isolate algal cells possessing high lipid content. © 2009 The Society for Biotechnology, Japan.

Shilova I.N.,University of California at Santa Cruz | Robidart J.C.,University of California at Santa Cruz | James Tripp H.,U.S. Department of Energy | Turk-Kubo K.,University of California at Santa Cruz | And 11 more authors.
ISME Journal | Year: 2014

Metagenomic approaches have revealed unprecedented genetic diversity within microbial communities across vast expanses of the world's oceans. Linking this genetic diversity with key metabolic and cellular activities of microbial assemblages is a fundamental challenge. Here we report on a collaborative effort to design MicroTOOLs (Microbiological Targets for Ocean Observing Laboratories), a high-density oligonucleotide microarray that targets functional genes of diverse taxa in pelagic and coastal marine microbial communities. MicroTOOLs integrates nucleotide sequence information from disparate data types: genomes, PCR-amplicons, metagenomes, and metatranscriptomes. It targets 19 400 unique sequences over 145 different genes that are relevant to stress responses and microbial metabolism across the three domains of life and viruses. MicroTOOLs was used in a proof-of-concept experiment that compared the functional responses of microbial communities following Fe and P enrichments of surface water samples from the North Pacific Subtropical Gyre. We detected transcription of 68% of the gene targets across major taxonomic groups, and the pattern of transcription indicated relief from Fe limitation and transition to N limitation in some taxa. Prochlorococcus (eHLI), Synechococcus (sub-cluster 5.3) and Alphaproteobacteria SAR11 clade (HIMB59) showed the strongest responses to the Fe enrichment. In addition, members of uncharacterized lineages also responded. The MicroTOOLs microarray provides a robust tool for comprehensive characterization of major functional groups of microbes in the open ocean, and the design can be easily amended for specific environments and research questions. © 2014 International Society for Microbial Ecology All rights reserved.

Thompson A.W.,Institute for Systems Biology | Crow M.J.,Advanced Cytometry Group | Wadey B.,Advanced Cytometry Group | Arens C.,Institute for Systems Biology | And 9 more authors.
Journal of Microbiological Methods | Year: 2015

A high speed flow cytometric cell sorter was modified to maintain a controlled anaerobic environment. This technology enabled coupling of the precise high-throughput analytical and cell separation capabilities of flow cytometry to the assessment of cell viability of evolved lineages of obligate anaerobic organisms from cocultures. © 2015 .

Kormelink T.G.,University Utrecht | Arkesteijn G.J.A.,University Utrecht | Nauwelaers F.A.,BD Biosciences Europe | van den Engh G.,Advanced Cytometry Group | And 3 more authors.
Cytometry Part A | Year: 2016

Submicron-sized vesicles released by cells are increasingly recognized for their role in intercellular communication and as biomarkers of disease. Methods for high-throughput, multi-parameter analysis of such extracellular vesicles (EVs) are crucial to further investigate their diversity and function. We recently developed a high-resolution flow cytometry-based method (using a modified BD Influx) for quantitative and qualitative analysis of EVs. The fact that the majority of EVs is <200 nm in size requires special attention with relation to specific conditions of the flow cytometer, as well as sample concentration and event rate. In this study, we investigated how (too) high particle concentrations affect high-resolution flow cytometry-based particle quantification and characterization. Increasing concentrations of submicron-sized particles (beads, liposomes, and EVs) were measured to identify coincidence and swarm effects, caused by the concurrent presence of multiple particles in the measuring spot. As a result, we demonstrate that analysis of highly concentrated samples resulted in an underestimation of the number of particles and an interdependent overestimation of light scattering and fluorescence signals. On the basis of this knowledge, and by varying nozzle size and sheath pressure, we developed a strategy for high-resolution flow cytometric sorting of submicron-sized particles. Using the adapted sort settings, subsets of EVs differentially labeled with two fluorescent antibodies could be sorted to high purity. Moreover, sufficient numbers of EVs could be sorted for subsequent analysis by western blotting. In conclusion, swarm effects that occur when measuring high particle concentrations severely hamper EV quantification and characterization. These effects can be easily overlooked without including proper controls (e.g., sample dilution series) or tools (e.g., oscilloscope). Providing that the event rate is well controlled, the sorting strategy we propose here indicates that high-resolution flow cytometric sorting of different EV subsets is feasible. © 2015 International Society for Advancement of Cytometry.

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