Tube rotator

EVs were isolated from cell culture media according to the NBI method [7 (link)]. Briefly, cells were grown until at least 70% confluence in complete medium. Then, 24 h before performing the isolation, cells were washed twice with PBS 1X, and medium was changed to serum-free medium. Cell culture medium was then collected and centrifuged at 2800× g for 10 min at RT to remove potential cellular debris. To the resulting supernatant, homogenised nickel-functionalized agarose beads [7 (link)] were added at a concentration of 25 μL of beads per 1 mL of sample. Samples were incubated for 30 min with gentle rotation (9–12 rotations/minute) using a VWR tube rotator to prevent sedimentation of beads and then centrifuged at 600–800× g for 2 min to sediment the EV-containing bead pellet. Then 1.2 X elution buffer was freshly prepared by diluting 5X Solution A (PBS 1X + 16 mM EDTA) and 5X Solution B (PBS 1X + 10 mM NaCl + 225 μM citric acid) in 0.22 μm-filtered PBS 1X. Two-bead volumes of elution buffer were added to the EV-containing bead pellet, and the elution was completed by incubating the samples in a thermomixer at 28 °C for 10 min at 600× g. After a final centrifugation step at 1800× g for 1 min at RT, EVs were collected from the supernatant. EV concentration and size were evaluated using a Nanosight instrument.

Fluorescence microscopy was used to investigate free sterol content and its cellular distribution by Filipin III staining (final concentration of 0.5 μg ml⁻¹) and neutral lipid content by Nile Red staining (final concentration of 2.5 μg ml⁻¹). For that, yeast cells from the parental and pdr18Δ strains harvested from cultivation at 30°C and 40°C were re‐suspended in PBS buffer to a final OD_600nm of 0.5. Cells were then incubated with each probe for 20 min, in the dark, in a tube rotator (VWR) and washed twice with PBS buffer. For cell‐to‐cell analysis, fluorescence was examined with an Axioplan microscope equipped with adequate epifluorescence interface filters, obtained from Zeiss. Fluorescence emission was collected with a coupled device camera (Axiocam 503 colour; Zeiss), and the images were analysed with ZEN 2 Microscope Software (Zeiss). The exposure time was kept constant among experiments for each probe, and intensity measurements were background‐corrected. For total quantification of the population's fluorescence, cell suspensions stained with either Filipin III or Nile Red were standardized to OD_600nm of 0.5 and the fluorescence was measured in a Filtermax F5 Microplate Reader (Ex/Em of 360/465 nm for Filipin III and 530/625 nm for Nile Red). Results were obtained from three independent biological replicates.

At 3 days post injection, ~ 30 midguts were dissected from the surviving females in 1x PBS and placed into 1.5 mL microcentrifuge tubes. A 30-min incubation in 10 μM ZnMP at room temperature was performed in aluminum foil covered microcentrifuge tubes placed on a tube rotator (VWR). Once the ZnMP incubation and wash steps were finished, treated midguts were placed on microscope slides in 1xPBS sectioned off with an ImmEdge™ hydrophobic barrier pen (Vector Laboratories). Images were taken of each midgut at 16x magnification with the DsRED filter of the Leica M165 FC stereomicroscope equipped with a DFC3000C digital camera. The resulting images were analyzed by ImageJ [51 (link)].

The whole-blood infection assay was performed as described [24 (link)]. Six chickens per line (in total 12 animals) were used. Blood samples from two to four animals (one or two from each line) were tested on the same day. Briefly, peripheral blood was drawn by wing venipuncture (Vena ulnaris) into commercial hirudin-coated syringes (S-Monovette®, 2.7 mL Hirudin, Sarstedt, Germany). Hirudin was chosen as anticoagulant as it was previously shown to have no effect on thrombocytes and complement activation [24 (link), 26 (link)]. The number of 10⁶ GFP-expressing microbial cells was added to 1 mL of blood. Samples were incubated at 41 °C, 5% CO₂ under constant rotation (20 rpm, Tube rotator, VWR, Darmstadt, Germany, product number VWRI444-0500) for 240 min. Every 30 min, a volume of blood was taken for flow-cytometric analysis (20 µL), determining the colony forming units of the pathogens (cfu, 10 µL) and for establishing the transcription levels of selected immune-related genes by RT-PCR (50 µL; 0, 30, 90, 150 and 240 min after inoculation of bacteria).