Andrade C et al. (2021), Characterization of Coelomic Fluid Cell Types i...

ECB-ART-49249

Front Immunol 2021 Jan 01;12:641664. doi: 10.3389/fimmu.2021.641664.

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Characterization of Coelomic Fluid Cell Types in the Starfish Marthasterias glacialis Using a Flow Cytometry/Imaging Combined Approach.

Andrade C , Oliveira B , Guatelli S , Martinez P , Simões B , Bispo C , Ferrario C , Bonasoro F , Rino J , Sugni M , Gardner R , Zilhão R , Coelho AV .

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Coelomocytes is the generic name for a collection of cellular morphotypes, present in many coelomate animals, and highly variable among echinoderm classes. The roles attributed to the major types of these free circulating cells present in the coelomic fluid of echinoderms include immune response, phagocytic digestion and clotting. Our main aim in this study was to characterize coelomocytes found in the coelomic fluid of Marthasterias glacialis (class Asteroidea) by using a combination of flow cytometry (FC), imaging flow cytometry (IFC) and fluorescence plus transmission electron microscopy (TEM). Two coelomocyte populations (P1 and P2) identified through flow cytometry were subsequently studied in terms of abundance, morphology, ultrastructure, cell viability and cell cycle profiles. Ultrastructurally, P2 diploid cells were present as two main morphotypes, similar to phagocytes and vertebrate thrombocytes, whereas the smaller P1 cellular population was characterized by low mitotic activity, a relatively undifferentiated cytotype and a high nucleus/cytoplasm ratio. In the present study we could not rule out possible similarities between haploid P1 cells and stem-cell types in other animals. Additionally, we report the presence of two other morphotypes in P2 that could only be detected by fluorescence microscopy, as well as a morphotype revealed via combined microscopy/FC. This integrative experimental workflow combined cells physical separation with different microscopic image capture technologies, enabling us to better tackle the characterization of the heterogeneous composition of coelomocytes populations.

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	Figure 1. Flow cytometry (FC) and imaging flow cytometry (IFC) analysis of circulating coelomocytes. Coelomocytes were stained with DRAQ5. (A) Representative FC dot plot of coelomocytes with cell populations gated. (B) Overlap of FC dot plots showing representative forward and side scatter properties of P1 (red) and P2 (blue) cell populations. (C) Overlap of P1 (red) and P2 (blue) FC cell populations histograms showing their median fluorescence intensity (MFI) in the DRAQ5 (660/20 BP) channel. (D) Representative IFC dot plot of coelomocytes with cell populations gated. (E) Overlap of IFC dot plots showing representative area and side scatter properties of P1 (red) and P2 (blue) cell populations. Insets show two examples of cells imaged in each population; scale bar: 7 μm. (F) Overlap of P1 (red) and P2 (blue) IFC cell populations histograms showing their median fluorescence intensity (MFI) in the DRAQ5 (642-745 nm) channel.
	Figure 2. Flow cytometric analysis of circulating coelomocytes viability. Cells were stained with DRAQ5 and DAPI. (A) Representative dot plot with P1 and P2 cell populations gated. (B) A quadrant plot of coelomocytes showing DAPI+ DRAQ5+ (dead) and DRAQ5+ DAPI- (alive) coelomocytes. (C) A dot plot showing that in the living cells quadrant, these belong mainly to the P2 cell population. (D) A dot plot showing that cells in the dead cells’ quadrant belong mainly to P1 cell population.
	Figure 3. Flow cytometric cell cycle histograms. Cells were fixed with 70% ethanol and stained with PI. (A) Representative histogram of P1 (red) and P2 (blue) singlets in the cell cycle, showing that for P1 most of the cells are in G0/G1 phase. (B) P2 cell distribution in G0/G1 (blue, 92%), S (yellow, 5%), or G2/M (green, 2%) phases was determined using the Dean Jett Fox fitting algorithm of FlowJo software (version 10.7), after excluding cell debris and aggregates. PI fluorescence is proportional to DNA content.
	Figure 4. Imaging flow cytometry morphologic analysis of circulating coelomocytes. (A) IFC dot plots showing cell area and FM 4-64 intensity values of P1 (red) and P2 (blue) cell populations. (B, C) Brightfield (BF) and FM4-64 fluorescence IFC images for the several morphotypes detected within each coelomocytes population, P1 (B) and P2 (C). Coelomocytes membranes were stained with FM 4-64 (red). B1. P1 cells displaying a less heterogenous brightfield image with the nucleus occupying the majority of the cell area (Figure 5A3) B2. P1 cells with a heavily granulated cytoplasm displaying a smaller nucleus to cell ratio (Figures 5A1, A2); C1. P2 regular; C2. P2 petaloid C3. P2 filopodial coelomocytes; C4. Big granulated cell.
	Figure 5. Characterization of coelomocytes morphologies by fluorescence microscopy. (A) Two morphotypes were identified within the P1 cell population: cells with a lower nucleus to cytoplasm ratio (1; area: 78 μm2 and 2. area: 26 μm2) and cells with a nucleus occupying most of the cell space (3; area: 39 μm2). Three morphotypes were observed within the P2 cell population: Regular (4; area: 130 μm2, 5; area: 354 μm2 and 6; area: 430 μm2), Petaloid (7; area: 561 μm2) and Filopodial (8; area: 463 μm2). (B) Representatives of novel P2 cell morphologies (1; area: 167 μm2 and 2. area: 171 μm2).
	Figure 6. Coelomocytes morphology by transmission electron microscopy. (A) Thrombocyte-like cell (tc) full of roundish electron-lucent vesicles (arrowheads) and with a cell process forming a loop (asterisk). (B) Macrophage-like cell (mc) showing few electron-lucent vesicles (black arrowhead), mitochondria (white arrowhead) and few short cytoplasmic extensions (arrow). A well-developed Golgi apparatus is visible in the top right inset. (C) Slightly undifferentiated cell with a high nucleus/cytoplasm ratio and several mitochondria (white arrowheads). (D) Aggregate of coelomocytes: both tc and mc possess cytoplasmic processes intermingled with each other (arrows). mc, macrophage-like cell; n, nucleus; tc, thrombocyte-like cell. Scale bars: A, B=2 µm; C=0.5 µm; D=5 µm.

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