50 ml plastic tubes

Each milk sample was analyzed for fat, protein, lactose, true protein and casein using a Bentley DairySpec FT (Technopath Distribution, Co. Tipperary, Ireland). Samples were heated to ~40 °C in 50 mL plastic tubes (Sarstedt Ltd., Wexford, Ireland) before analysis. Results were expressed as the average of 2 replicates.

HAP1 cells of each genotype were grown in T175 cm² flasks (Sarstedt) in 10 mM MgCl₂ supplemented cell culture medium as described above. At ~50% confluence the medium was replaced with fresh medium without 10 mM MgCl₂ and the cells were cultured for additional 24 hr. Next, cells were washed with PBS, disaggregated by trypsinization and collected in 50 ml plastic tubes (Sarstedt). After centrifugation (3 min, 1000 rpm), the cell pellet was resuspended in 1 ml PBS and passed to a fresh 1.5 ml tube. The cell suspension was centrifuged (3 min, 3500 rpm), supernatant was completely removed and the cell pellet was dried overnight at 70°C. The dried cell pellet was analysed by ICP-MS in ALS Scandinavia (Sweden). The experiment was repeated four times.

Roots were sampled and analyzed via ink staining [59 ]. The roots were cut into small pieces and placed in 50-ML plastic tubes (Sarstedt, Nümbrecht, Germany). Twenty-five mL of KOH 10% was added to the roots before incubation at 70 °C in a water bath for 45 min. The KOH was then removed, and roots were washed with HCl 1%. The staining step consisted of adding 25 mL of ink 2% (Parker Pen Company, Nantes, France) in HCl 1%. The tubes were then placed at 70 °C in a water bath for 30 min. The roots were rinsed and stored in deionized water before analysis. The total colonization rate was quantified using the gridline intersect method [60 (link)]. Positive counts for AM colonization included the presence of vesicles or arbuscules or typical mycelium within the roots.