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Sci Rep
2020 Jan 24;101:1149. doi: 10.1038/s41598-019-57387-7.
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Autofluorescence mediated red spherulocyte sorting provides insights into the source of spinochromes in sea urchins.
Hira J
,
Wolfson D
,
Andersen AJC
,
Haug T
,
Stensvåg K
.
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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 high-resolution 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.
Figure 1. Brightfield and deconvolved fluorescence images of RSCs. (A) Bright field and (B) fluorescence images of a mix population of CSCs and RSCs at time 0 min. (C) Bright field and (D) fluorescence images after one hour of continuous fluorescence imaging of the same field of view. (E) Close-up fluorescence images at representative time points showing morphological changes in the cell highlighted in (B). Scale bar 10 µm (A–D) and 5 µm (E).
Figure 2. Flow cytometry based autofluorescent (AF) RSCs sorting. Here, FCS Express 6.06.0014 (De Novo Software) generated two-parameter density plot shows the density of cells at a given position represented by different colours. (A) Sampling of coelomocytes and RSCs enrichment. The density plot represents; (B) Forward/Scatter profile of live unstained coelomocyte. (C) Microscopic image of RSCs and other cells before sorting. (D) Gating of singlets allowing discrimination of doublets and (E) Gating of distinct fluorescent RSCs population represented by the red box. (F) Histogram of fluorescent RSCs population. The histogram marker presents the selective population to be sorted. (G) Microscopic image of pure AF-RSCs after sorting. Microscopic image scale bar 50 µm.
Figure 3. UHPLC-MS analysis of MPCs and RSCs H2O extracts. (A) Base peak chromatogram of MPCs H2O extract. (B) Base peak chromatogram of RSCs H2O extract depicting the tentative identification of previously unreported sulphate derivatives of spinochromes and suspected previously reported spinochromes. I: Sulphate derivative of spinochrome E isomer 1. II: Sulphate derivative of spinochrome E isomer 2. III: Suspected sulphate derivative of spinochrome D. IV: Suspected sulphate derivative of spinochrome dimer (m/z 614.9926). V: Suspected sulphate derivative of spinochrome C. VI: Suspected sulphate derivative of spinochrome related dimer ethylidene-6, 60-bis (2,3,7-trihydroxynaphthazarin) (m/z 580.9871). All tentatively identified metabolites were absent in extracts of the MPCs substrate, with the exception of trace amounts of sul-SpinD. Scale bar 50 µm.
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