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Viable cell sorting of dinoflagellates by multiparametric flow cytometry.

ABSTRACT: Electronic cell sorting for isolation and culture of dinoflagellates and other marine eukaryotic phytoplankton was compared to the traditional method of manually picking cells using a micropipette. Trauma to electronically sorted cells was not a limiting factor, as fragile dinoflagellates, such as Karenia brevis (Dinophyceae), survived electronic cell sorting to yield viable cells. The rate of successful isolation of large-scale (> 4 litres) cultures was higher for manual picking than for electronic cell sorting (2% vs 0.5%, respectively). However, manual picking of cells is more labor intensive and time consuming. Most manually isolated cells required repicking, as the cultures were determined not to be unialgal after a single round of isolation; whereas, no cultures obtained in this study from electronic single-cell sorting required resorting. A broad flow cytometric gating logic was employed to enhance species diversity. The percentages of unique genotypes produced by manual picking or electronic cell sorting were similar (57% vs 54%, respectively), and each approach produced a variety of dinoflagellate or raphidophyte genera. Alternatively, a highly restrictive gating logic was successfully used to target K. brevis from a natural bloom sample. Direct electronic single-cell sorting was more successful than utilizing a pre-enrichment sort followed by electronic single-cell sorting. The appropriate recovery medium may enhance the rate of successful isolations. Seventy percent of isolated cells were recovered in a new medium (RE) reported here, which was optimized for axenic dinoflagellate cultures. The greatest limiting factor to the throughput of electronic cell sorting is the need for manual postsort culture maintenance and assessment of the large number of isolated cells. However, when combined with newly developed automated methods for growth screening, electronic single-cell sorting has the potential to accelerate the discovery of new algal strains.

SUBMITTER: Sinigalliano CD

PROVIDER: S-EPMC2841404 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature

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Viable cell sorting of dinoflagellates by multiparametric flow cytometry.

Sinigalliano Christopher D CD Winshell Jamie J Guerrero Maria A MA Scorzetti Gloria G Fell Jack W JW Eaton Richard W RW Brand Larry L Rein Kathleen S KS

Phycologia 20090701 4

Electronic cell sorting for isolation and culture of dinoflagellates and other marine eukaryotic phytoplankton was compared to the traditional method of manually picking cells using a micropipette. Trauma to electronically sorted cells was not a limiting factor, as fragile dinoflagellates, such as Karenia brevis (Dinophyceae), survived electronic cell sorting to yield viable cells. The rate of successful isolation of large-scale (> 4 litres) cultures was higher for manual picking than for electr ...[more]

PMID: 20305733

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Project description:Simple Summary The tumor microenvironment in breast cancer plays important roles in tumor development and treatment response, giving important information critical for disease management. Today, an analysis of the tumor microenvironment is included in routine histopathologic reporting for practical clinical application. This manuscript aimed to deepen the study of the tumor microenvironment, analyzing the immune cells in breast tumoral and benign pathologies. Indeed, using a deep immunophenotyping approach by flow cytometry, we have studied the immune cells at the level of breast tissues, identifying different immunophenotyping that could be useful in the diagnosis and follow up of breast pathologies. As possible targets are continually being discovered in the tumor microenvironment, a future approach to breast cancer diagnosis and therapy could likely combine cancer cell elimination and tumor microenvironment modulation. Abstract Immune cell components are able to infiltrate tumor tissues, and different reports described the presence of infiltrating immune cells (TILs) in several types of solid tumors, including breast cancer. The primary immune cell component cells are reported as a lymphocyte population mainly comprising the cytotoxic (CD8+) T cells, with varying proportions of helper (CD4+) T cells and CD19+ B cells, and rarely NK cells. In clinical practice, an expert pathologist commonly detects TILs areas in hematoxylin and eosin (H&E)-stained histological slides via light microscopy. Moreover, other more in-depth approaches could be used to better define the immunological component associated with tumor tissues. Using a multiparametric flow cytometry approach, we have studied the immune cells obtained from breast tumor tissues compared to benign breast pathologies. A detailed evaluation of immune cell components was performed on 15 and 14 biopsies obtained from breast cancer and fibroadenoma subjects, respectively. The percentage of tumor-infiltrating T lymphocytes was significantly higher in breast cancer patients compared to patients with fibroadenoma. Infiltrating helper T lymphocytes were increased in the case of malignant breast lesions, while cytotoxic T lymphocytes disclosed an opposite trend. In addition, our data suggest that the synergistic effect of the presence/activation of NK cells and NKT cells, in line with the data in the literature, determines the dampening of the immune response. Moreover, the lymphocyte-to-monocyte ratio was calculated and was completely altered in patients with breast cancer. Our approach could be a potent prognostic factor to be used in diagnostic/therapeutic purposes for the improvement of breast cancer patients’ management.

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Viable cell sorting of dinoflagellates by multiparametric flow cytometry.

Publications

Viable cell sorting of dinoflagellates by multiparametric flow cytometry.

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