Shandon formal fixx

Cell migration was analyzed by a Boyden chamber assay utilizing ThinCert® transwell polyethylene terephthalate (PET) membrane supports (8 µm pore size; Greiner Bio-One, St. Gallen, Switzerland) as described (Asparuhova et al., 2021 (link)). After 24 h of starvation, 3 × 10⁴ cells were cultured in the top insert chamber with 200 µl 0% FCS/DMEM. Each of the collagen matrices was placed in the low chamber with 800 µl 10% FCS/DMEM. Cells were allowed to migrate across the capillary pore PET membrane for 18 h at 37°C before fixation in Shandon™ Formal-Fixx™ (ThermoFisher Scientific), and staining in 0.1% crystal violet solution (Sigma). Images of duplicate inserts were acquired on an Olympus CKX41 microscope using a ProgResCT3 camera. Migration was quantified by using the ImageJ software (Schneider et al., 2012 (link)) as described (Gurbuz et al., 2014 (link)). Data represent means ± SD from three independent experiments performed with each of the two cell lines.

At the end of the study (95 d of age), calves were slaughtered to collect digesta samples and segments of the rumen and the small intestine for microbiota analysis and morphometry analysis, respectively. The slaughtering involved stunning with a captive bolt pistol followed by immediate exsanguination at the livestock infectious disease isolation facility of Iowa State University (Ames, IA, USA). About 300 g of rumen content of the ventral sac and about 50 g of digesta from the jejunum were retrieved and placed immediately in dry ice until transfer to a −80 °C freezer. The jejunum was specifically chosen for the microbiome analysis (described below), as both digesta and mucosa of the jejunum were shown to have a greater and similar density of bacteria (numbers per gram wet weight) compared to those of the duodenum and ileum in cattle, respectively [21 (link)]. Additionally, 4.0–6.0 cm² segments of the rumen (ventral sac), and 2–3 cm long segments of the duodenum, jejunum (15 and 80 cm distal to pyloric sphincter, respectively), and ileum (15 cm proximal to ileocecal junction) were obtained. All samples were flushed with saline to remove any digesta and were fixed in 10% neutral-buffered formaldehyde (Shandon Formal-Fixx^®, Thermo Scientific, Waltham, MA, USA) for the morphometric analysis described below.