Matrix d fastprep tubes

Acute colitis was induced at day 0 by adding 3% (w/v) DSS (36,000–50,000 Da; MP Biomedicals, Santa Ana, CA, USA) to their drinking water. DSS was fed to the mice of the colitis groups from day 0 to day 7. Water-fed mice served as control. On day 2, BM-Eos were suspended in RPMI-1640 at 1 × 10⁷ cells/ ml and 200 μL of this cell suspension per mouse (= 2 × 10⁶ cells) were injected intravenously into the tail veins of IL-5-deficient mice (Figure 1). WT or IL-5-deficient mice without i.v. injection of cells served as DSS-fed controls. On day 7, mice were sacrificed by exsanguination after sedation with CO₂, the sera were collected and the cola and ceca were removed. The lengths of cola and ceca were measured and the collected tissues were washed with PBS to remove remaining feces. One third of each colon was fixed in 4% (v/v) formaldehyde in PBS, embedded in paraffin, and further processed for hematoxylin/eosin (H&E) staining. Another third of each colon was stored in RNAlater for qPCR analyses, the remaining third of each colon was snap frozen in 2 mL Matrix D FastPrep® tubes (MP Biomedicals, Santa Ana, CA, USA). Mesenteric lymph nodes were removed and a lymphocyte enriched single cell suspension was generated by passing the tissue through a sterile 100 μm mesh with 5 mL RMPI-1640 medium.

Mesenteric lymph node cells were seeded at 1 × 10⁶ cells/ml 200 μl/well in RPMI-1640 + 10% (v/v) FCS, 50 μM ß-MeSH on a 96 well-plate, and incubated at 37°C, 5% (v/ v) CO₂ for 24 h in the absence or presence of CD3-antibody (EXBIO Praha, Vestec u Prahy, Czech Republic). After the incubation period, cell-free supernatants were generated by centrifugation at 300 g for 10 min at 4°C. Colon tissues were homogenized either for cytokine measurements in ice cold PBS + 1x protease inhibitor cocktail (Cell Signaling Technology, Frankfurt, Germany) or for RNA extraction in lysis buffer RA1 (Macherey Nagel, Düren, Germany) supplemented with 150 mM ß-MeSH using the FastPrep® 24 homogenizer and 2 mL Matrix D FastPrep® tubes (both from MP Biomedicals, Santa Ana, CA, USA) according to the manufacturer's instructions for intestinal tissue.