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Adherent Intestinal Cells from Atlantic Salmon Show Phagocytic Ability and Express Macrophage Markers

ABSTRACT: Atlantic salmon (average 550g) were used in this study. The fish were starved for 24 h and were sacrificed with an overdose of MS-222. Then head kidney (HK) and distal intestine (DI) samples were collected. Immune cells from HK and DI were isolated and cultured at 12°C in Leibovitz’s L-15 Medium (L-15). Both HK and DI leukocytes were allowed to adhere on a petri dish with 2 mL L-15+ for 2 days at 12°C. After removing the supernatant containing non-adherent cells, the petri dish with the adherent cells was placed on ice for 10 min, and the cells were detached by washing three times with 1.5 mL ice-cold PBS supplemented with 5 mM EDTA. The cells obtained were centrifuged (500 × g, 5 min, 4°C) and re-suspended in 1 mL L-15+. Then, the cells were counted using a portable cell counter. Total RNA was extracted from adherent cells from HK or DI (< 500,000 cells). To compare the expression of marker genes between adherent cells from HK and DI, 12 libraries were prepared (6 replicates for each). Transcriptomic analysis revealed that adherent intestinal cells (AICs) expressed genes associated with macrophages, T cells, and endothelial cells. In addition, AIC had higher expression of TNF and TGFβ-related genes that can be linked to macrophages and T cells. Heatmap analysis of selected genes indicated that the adherent cells from the two organs had apparently higher expression of macrophage-related markers.

ORGANISM(S): Salmo salar

PROVIDER: GSE154142 | GEO | 2020/11/10

REPOSITORIES: GEO

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Project description:Single-cell RNA-seq data from the mouse Dorsomedial Hypothalamic Nucleus (DMH) was generated following a modified protocol from https://www.nature.com/articles/s41586-020-2821-8. Briefly, six 7.5 week old C57B6 mice (3 male, 3 female) were anaesthetized with isoflurane. Brains were rapidly extracted and dropped into ice-cold carbogenated NMDG-HEPES-ACSF. Brain sections (2 mm) containing DMH were cut with a razor blade on a stainless steel brain matrix (51392, Stoelting) and transferred to a dissection dish on ice containing NMDG-HEPES-ACSF. DMH was bilaterally microdissected in ~700·700·350 micron tissue block. Microdissected tissue was aggregated in a collection tube on ice containing NMDG-HEPES-ACSF and 30 μM actinomycin D. For enzymatic tissue digestion, NMDG-HEPES-ACSF was replaced by trehalose-HEPES-ACSF containing papain (50 U/ml; P3125, Sigma Aldrich, pre-activated with 2.5 mM cysteine and a 30-min incubation at 34 °C and supplemented with 0.5 mM EDTA) and 15 μM actinomycin D. Extracted DMH tissue was incubated at room temperature with gentle carbogenation for 55 min. During enzymatic digestion the tissue was pipetted periodically every 10 min. At the end of enzymatic digestion, the medium was replaced with 200μl of room temperature trehalose-HEPES-ACSF containing 3 mg/ml ovomucoid inhibitor (OI-BSA,Worthington)25 U/ml DNase I (90083, Thermo Scientific) and 15 μM actinomycin D and tissue was gently triturated into a uniform single-cell suspension with fire-polished glass Pasteur pipettes. The resulting cell suspension volume was brought up to 1 ml with trehalose-HEPES-ACSF with 3 mg/ml ovomucoid inhibitor and pipetted through a 40μm cell strainer. Single-cell suspension was centrifuged down at 300g for 5 min at 4 °C and the supernatant was replaced with 1 ml fresh ice-cold trehalose-HEPES-ACSF and the cell pellet was resuspended. Cells were pelleted again and resuspended in 100 μl of ice-cold resuspension-ACSF. Cell suspensions were kept on ice while cell densities were quantified with a haemocytometer and the final cell densities were verified to be in the range of 300–1,000 cells/μl. Cell suspension volumes estimated to retrieve ~10,000 single-cell transcriptomes were added to the 10x Genomics RT reaction mix and loaded to the 10X Next GEM Chip G (2000177, 10x Genomics) per the manufacturer’s protocol. We used the Chromium Next GEM Single Cell 3’ Reagents Kit v3.1 (1000128, 10x Genomics) and the Single Index Kit Set A (100213) to prepare Illumina sequencing libraries downstream of reverse transcription following the manufacturer’s protocol. Resulting scRNA-seq sequencing library was sequenced on an Illumina NovaSeq 6000 sequencers (paired-end 150). Sequencing data were aligned to optimized pre-mRNA reference transcriptome52 and digital gene-cell matrices were generated with the 10x Genomics Cell Ranger v.6.0.0 count pipeline. The resulting scRNA-seq data were analyzed in R (4.1.2) using Seurat (v.4.1.0.9007) as previously described51. Briefly, expression data were filtered to exclude cells with fewer than 1,000 unique transcripts as well as cells exhibiting more than 15% of mitochondrial transcripts. We followed the standard Seurat workflow to identify transcriptomic cell-types and assigned cel class identities based on canonical cell-type markers: Ndrg4 for neurons, Ntsr2 for astrocytes, Slco1c1 for endothelial cells, Fcer1g for microglia and Mag for oligodendrocytes. We digitally extracted neuronal clusters from our dataset resulting in expression data for 4587 DMH neurons that we used to establish an unbiased neuronal nomenclature for DMH.

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Adherent Intestinal Cells from Atlantic Salmon Show Phagocytic Ability and Express Macrophage Markers

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