Methods for collection, handling, and analysis of sea urchin coelomocytes

Smith, L. Courtney; Hawley, Teresa S.; Henson, Joan M.; Majeske, Audrey J.; Oren, Matan; Rosental, Benyamin

doi:10.1016/bs.mcb.2018.11.009

Cited by 32 publications

(37 citation statements)

References 59 publications

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“…These simple cytometric patterns are consistent with several reports (11), (19) that detect phagocytes as the most abundant, or even the only circulating coelomocyte type in asteroids. A higher cellular diversity was described later by Smith and collaborators (2019) (22) in Strongylocentrotus purpuratus. In that manuscript, and using FC, live cells were selected based on exclusion of propidium iodide.…”

Section: Coelomocyte Fractionation Using Flow Cytometrysupporting

confidence: 55%

“…Direct comparison of echinoid coelomocyte types identified by FC with those of Asteroidea is difficult given the known variability of coelomocyte types among echinoderm classes (7); (22) and by the diversity of protocols, for instance, the specific lectins-labelling method used by Liao and collaborators (2017) (26).…”

Section: Coelomocyte Fractionation Using Flow Cytometrymentioning

confidence: 99%

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A combined characterization of coelomic fluid cell types in the spiny starfishMarthasterias glacialis– inputs from flow cytometry and imaging

Oliveira

Guatelli

Martı́nez

et al. 2020

Preprint

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Coelomocytes is a generic name for a collection of cellular morphotypes, present in many coelomate animals, that has been reported as highly variable across echinoderm classes. The roles attributed to the major types of the free circulating cells present in the coelomic fluid of echinoderms include immune response, phagocytic digestion and clotting. The main aim of the present study is the thorough characterization of coelomocytes present in the coelomic fluid of Marthasterias glacialis (class Asteroidea) through the combined use of flow cytometry (FC) and fluorescence plus transmission electron microscopy. Two coelomocyte populations (here named P1 and P2) were identified by flow cytometry and subsequently studied in terms of abundance, morphology, ultrastructure, cell viability and cell cycle profiles. Ultrastructurally, P2 diploid cells showed two main morphotypes, similar to phagocytes and vertebrate thrombocytes, whereas the small P1 haploid cellular population was characterized by a low mitotic activity, relatively undifferentiated cytotype and a high nucleus/cytoplasm ratio. These last cells resemble stem-cell types present in other animals. P1 and P2 cells differ also in cell viability and cell cycle profiles. Additionally, two other morphotypes were only detected by fluorescence microscopy and a third one when using a combined microscopy/FC approach.

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Section: Coelomocyte Fractionation Using Flow Cytometrysupporting

confidence: 55%

Section: Coelomocyte Fractionation Using Flow Cytometrymentioning

confidence: 99%

A combined characterization of coelomic fluid cell types in the spiny starfishMarthasterias glacialis– inputs from flow cytometry and imaging

Oliveira

Guatelli

Martı́nez

et al. 2020

Preprint

View full text Add to dashboard Cite

show abstract

“…CF (500 μl) from adult sea urchins was withdrawn from the coelomic cavity using a 1 ml syringe with a 21 gauge needle and expelled directly into Ca-, Mg-free artificial sea water with 70 mM EDTA and 20 mM HEPES pH 7.4 [CMFSW-EH, see (44)] at a 1:1 ratio. Cells were filtered through gauze pads, pelleted for 5 min at 400 × g at 4°C and resuspended in staining medium (3.3X PBS with 20 mM HEPES pH = 7.4 and 1.5% fetal calf serum) according to Smith et al (45). Cells were incubated with a mix of three polyclonal rabbit antibodies against the SpTrf proteins as described (18) at a dilution of 1:100 in 100 μl staining medium for 30 min on ice followed by centrifugation at 400 × g for 5 min at 4°C and resuspension in staining medium.…”

Section: Methodsmentioning

confidence: 99%

“…Coelomocytes were sorted on a FACSAria flow cytometer (BD Biosciences) equipped with blue, red, and violet lasers as described (45). Dead cell counts were gated out based on their nuclear PI excitation at 488 nm and detection with a 585/42 nm band pass (BP) filter.…”

Section: Methodsmentioning

confidence: 99%

Individual Sea Urchin Coelomocytes Undergo Somatic Immune Gene Diversification

et al. 2019

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The adaptive immune response in jawed vertebrates is marked by the ability to diversify somatically specific immune receptor genes. Somatic recombination and hypermutation of gene segments are used to generate extensive repertoires of T and B cell receptors. In contrast, jawless vertebrates utilize a distinct diversification system based on copy choice to assemble their variable lymphocyte receptors. To date, very little evidence for somatic immune gene diversification has been reported in invertebrate species. Here we show that the SpTransformer ( SpTrf ; formerly Sp185/333 ) immune effector gene family members from individual coelomocytes from purple sea urchins undergo somatic diversification by means of gene deletions, duplications, and acquisitions of single nucleotide polymorphisms. While sperm cells from an individual sea urchin have identical SpTrf gene repertoires, single cells from two distinct coelomocyte subpopulations from the same sea urchin exhibit significant variation in the SpTrf gene repertoires. Moreover, the highly diverse gene sequences derived from single coelomocytes are all in-frame, suggesting that an unknown mechanism(s) driving these somatic changes involve stringent selection or correction processes for expression of productive SpTrf transcripts. Together, our findings infer somatic immune gene diversification strategy in an invertebrate.

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“…Previously, it has been reported that the sea urchins larvae and embryonic pigment cells emanate spectacular autofluorescence 38,39 . A recent study has shown that the RSCs of adult sea urchin emits a weak autofluorescence when excited with 633 nm in the far red channel using FACS and it was suggested that the method requires further optimization for efficient sorting of these cells using FACS 40 .…”

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confidence: 99%

Autofluorescence mediated red spherulocyte sorting provides insights into the source of spinochromes in sea urchins

Hira

Wolfson

Andersen

et al. 2020

Sci Rep

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Red spherule cells (RSCs) are considered one of the prime immune cells of sea urchins, but their detailed biological role during immune responses is not well elucidated. Lack of pure populations accounts for one of the major challenges of studying these cells. In this study, we have demonstrated that live RSCs exhibit strong, multi-colour autofluorescence distinct from other coelomocytes, and with the help of fluorescence-activated cell sorting (FACS), a pure population of live RSCs was successfully separated from other coelomocytes in the green sea urchin, Strongylocentrotus droebachiensis. This newly developed RSCs isolation method has allowed profiling of the naphthoquinone content in these cells. With the use of ultra high-performance liquid chromatography, UV absorption spectra, and highresolution tandem mass spectrometry, it was possible to identify sulphated derivatives of spinochrome C, D, E and spinochrome dimers, which suggests that the RSCs may play an important biological role in the biogenesis of naphthoquinone compounds and regulating their bioactivity.

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Methods for collection, handling, and analysis of sea urchin coelomocytes

Cited by 32 publications

References 59 publications

A combined characterization of coelomic fluid cell types in the spiny starfishMarthasterias glacialis– inputs from flow cytometry and imaging

A combined characterization of coelomic fluid cell types in the spiny starfishMarthasterias glacialis– inputs from flow cytometry and imaging

Individual Sea Urchin Coelomocytes Undergo Somatic Immune Gene Diversification

Autofluorescence mediated red spherulocyte sorting provides insights into the source of spinochromes in sea urchins

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