High precision coverslips

Barley metaphase chromosomes (Hordeum vulgare L. cv. Morex) were sorted according to Lysák et al. (1999 (link)). Briefly, a chromosome suspension was prepared from synchronized primary roots meristems. Chromosomes were DAPI-stained, immediately analyzed, and flow-sorted using a FACSAria II SORP flow cytometer and sorter (BD Bioscience, San Jose, CA, USA). Five thousand chromosomes were sorted into 15 μl of PRINS buffer supplemented with 2.5% sucrose (10 mM TRIS, 50 mM KCl, 2 mM MgCl₂.6H₂O, 2.5% sucrose; pH 8) onto high precision coverslips (Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany). Before immunolabeling, the coverslips were stored at −20 °C.

Co-cultivation of C. difficile with the human colon carcinoma cell line Caco-2 (ATCC No. HTB-37) was based on a previously described protocol (Natoli et al., 2012 (link)). Cells were routinely grown in Dublecco’s Modified Eagle’s Medium (DMEM, Gibco, Thermo Fisher Scientific) supplemented with 10% inactivated fetal calf serum (FCS) (Sigma Aldrich, Taufkirchen, Germany) and 1% MEM non-essential amino acids (Gibco) in a 5% CO₂ atmosphere at 37°C. Confluency occurred after 5–6 days of incubation. For the adhesion assay, propagated cells were seeded at 5 × 10⁴ per well onto pre-cleaned and poly-L-lysine-coated (PLL; Sigma, 1:10 (v/v) in deionized water) 10 or 18-mm-diameter high precision coverslips (Paul Marienfeld GmbH, Lauda-Königshof, Germany) in a 12-well tissue culture plate (TPP Techno Plastic Products AG, Trasadingen, Switzerland). The culture medium was changed every other day. Cells were used 7 days (almost confluent monolayers) after seeding. The state of polarization and morphological differentiation was monitored by scanning electron microscopy (SEM).

Glass coverslips and microfluidic chambers were coated with 1 mg/ml poly-L-lysine (Sigma #P-2636) in borate buffer (3.1 mg/ml boric acid, 4.75 mg/ml borax, pH 8.50) overnight at room temperature. The dishes were then subjected to a short rinse, a long rinse (1 h), and two short rinses with sterile water. Finally, DMEM (Life Technologies, 31966047) plus 10% horse serum (Life Technologies, 26050088) was added to the dishes and brought to 37°C. For STED imaging, high-precision coverslips (Marienfeld, 117580) were used.