Project description:Mus musculus macrophages were derived from bone marrow, and treated with the supernatant of of DYRK1B-deletion (multiple clones) or Wildtype mKpc4 cells.
Project description:Effect of LPA-Mixture (16:0, 18:0 18:1 18:2, 20:4) and LPAR2 specific (H2L5186303) or LPAR1 specific (Ro6842262) Inh. in OC91s cells.
Project description:RNA-seq of differentiated organoids (day 7) from a patient with an AGR2 mutation, patients with ulcerative colitis, or non-IBD controls. After 6 days differentiation, organoids were treated for 24 hours with Tunicamycin (T7765, Sigma Aldrich,1 µg/ml) or vehicle control (0.1 % DMSO).
Project description:Patient-derived intestinal organoids provide an excellent tool to unravel mechanisms underlying ulcerative colitis (UC). Fresh biopsies, to isolate crypts and culture organoids, were obtained from both inflamed and non-inflamed regions from eight patients with active UC (Mayo endoscopic subscore ≥2), and from eight non-IBD controls.To address the inflammatory character of ex vivo organoids, we compared the transcriptome of biopsies, crypts and organoids derived from inflamed, and non-inflamed regions and aimed to (re-)induce inflammation ex vivo.
Project description:Spheroids, near spherical multicellular aggregates, are one of the most common types of threedimensional (3D) cell cultures. Despite decades of implementation of spheroid technology in various fields of life science and medical research, no minimal information (MI) guidelines are available to cope with heterogeneity and to stimulate transparency. To cope with this unmet need, we assembled an international consortium to develop the MISpheroID knowledgebase (https://www.mispheroid.org) and interrogation revealed heterogeneity and lack of transparency in published spheroid-related experiments. This steered us to empirically evaluate the impact of cell line, culture medium type, spheroid formation method and spheroid size on complementary spheroid metrics (RNA fingerprints, presence of cell death, ATP content, glucose to lactate conversion, secreted protein signatures, circularity, size and cancer therapy response). We measured media-induced transcriptional variation in lung cancer (A549), colorectal cancer (HCT116), ovarium cancer (SKOV3) and glioblastoma (U87MG) spheroids using RNA sequencing (RNA-seq). These spheroids were formed in ultra-low attachment plates and cultured in 6 different media types (DMEM high glucose, DMEM/F12, RPMI1640, DMEM low glucose, EMEM and MEM) for 5 (HCT116) or 7 (A549, SKOV3 and U87MG) days. RNA extraction was performed on 2 spheroids per condition using the miRNeasy micro kit (217084, Qiagen, Hilden, Germany). RNA-sequencing libraries were prepared from purified RNA using the QuantSeq 3' mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen, Vienna, Austria) according to the manufacturer's instructions, using 27.5ng of RNA that was DNase treated using HL-dsDNA (Arcticzymes, TromsØ, Norway). The individual libraries were quantified by qPCR using the KAPA Library Quantification Kit (Roche, Pleasanton, CA, US) and equimolarly pooled. The pool concentration was measured with Qubit and 1.4pM with 1% PhiX was sequenced on a NextSeq 500 (Illumina, San Diego, CA, US) using a high-output 1x75 run. Reads were mapped to the human genome using Tophat and gene expression counts were generated using HTSeq. This data was used to perform Principal Component Analysis (PCA) and Gene Set Enrichment Analysis (GSEA).
Project description:The effects of maternal microbiota on the fetal development was investigated by comparing tissues of fetuses from germ-free (GF) and normal (SPF) murine dams using RNA-seq and non-targeted metabolomics (for metabolomics data, see: https://bmcmicrobiol.biomedcentral.com/articles/10.1186/s12866-022-02457-6). For RNA-seq, two E18.5 fetuses were collected from 6 GF dams and 6 SPF dams, and transcriptomes analyzed by QuantSeq in whole intestine, brain and placenta.