15 ml centrifuge tube

Before digestion, all samples were briefly centrifuged at 2400 × g (Heraeus Pico 17 Centrifuge; Thermo Fisher Scientific, Waltham, MA, United States) to ensure that the tissue sat at the bottom of the tubes. Concentrated nitric acid (A509 Trace Metal Grade; Fisher, Loughborough, United Kingdom) and 5% Agilent Internal Standard mixture (5183-4681; Agilent Technologies, Cheadle, United Kingdom) were used to make the tissue digestion mixture. Calibration standards were prepared to the appropriate dilutions (Supplementary Table 4) using an Environmental Calibration Standard Mixture (Agilent 5189-4688) and 2% nitric acid digestion mix. For both wet- and dry-weight analysis, 200 mL of digestion mix was added to each sample including two empty 2-mL microcentrifuge tubes for use as digestion blanks. All microcentrifuge tube lids were punctured using a septum remover to prevent pressure build up before being transferred into a Dri-Block DB3 heater (Techne, Staffordshire, United Kingdom) at room temperature. The temperature was set to 60°C for 30 min and then increased to 100°C for a further 3.5 h. After the digestion procedure, 100 μL of each sample or digestion blank was added to 5 mL of LC/MS grade water in 15-mL centrifuge tubes (Greiner). Samples were retained at room temperature pending ICP-MS analysis.

Frankia cells acclimated to NH₃-deficient (N-) conditions were prepared as described for ARA measurements. A sample of the culture was taken 7 d after being transferred to BAP-TN- medium, and vesicles and hyphae were observed by a differential interference contrast (DIC) microscope (Eclipse TE2000-U; Nikon, Tokyo, Japan) and phase-contrast microscope (IX-70; Olympus, Tokyo, Japan). The number of vesicles was counted according to the method described previously (27 ). Briefly, we placed 2 mL of the culture in a 15-mL centrifuge tube (Greiner) and fragmented hyphae using the SoniMix ultrasonic homogenizer UX-050 (Mitsui Electric) with an output power setting of 38% for ~30 s. We applied the cell suspension to the interspace between a microscope slide and coverslip, the height of which was set to 0.01 mm using carbon steel tape (MonotaRO, Amagasaki, Japan). The prepared slide was observed using the DIC microscope and 5 to 9 pictures (area=0.345 mm² and volume=0.00345 mm³) were taken. The number of vesicles in each picture were counted and normalized by the protein content of the culture. Total protein concentrations were measured with the procedure described above.

Once the mouse had been killed, its right footpad (for mycolactone amounts assessment at different time after injection and for assessment of mycolactone diffusion) or tail (for inoculation with M. ulcerans) was removed, cut into small pieces and placed in a 15 mL centrifuge tube (Greiner Bio-one, Les Ulis, France). For both experiments, 2 mL methanol was added and the tissues were ground with a Potter-Elvehjem pestle (Dutscher Scientific, Issy-les-Moulineaux, France; 045080). The suspension was transferred to a 15 mL solvent-resistant centrifuge tube (VWR, Fontenay-sous-bois; 525-0401) and 4 mL chloroform was added. The mixture was then incubated overnight, in the dark, with shaking. Folch’s extraction was completed by adding 600 μL distilled water and shaking the mixture vigorously. The mixture was then centrifuged at 4000 × g for 10 min, to separate the aqueous and organic phases. The organic phase, containing the lipids, was transferred to a glass tube and allowed to dry in a centrifugal evaporator. The remaining material was recovered in 200 μL ice-cold acetone for phospholipid precipitation. The suspension was centrifuged for 10 min at 4000 × g. The supernatant was collected and stored at -20°C, in the dark, in amber glass vials, until use.

We followed the FF method as described previously (13 (link)). Mutagenized hyphae cultured in CB medium (2 mL) for a few weeks were transferred to a 15-mL centrifuge tube (Greiner, Tokyo, Japan) and fragmented using the SoniMix ultrasonic homogenizer UX-050 (Mitsui Electric, Chiba, Japan) with an output power setting of 50% for 10 s. Five hundred microliters of homogenized hyphae were transferred to Ultrafree centrifugal filter units (5-μm pore; Millipore, Billerica, MA, USA) and centrifuged at 12,000×g for 1 min. The filtrate was spread onto solid CB medium and incubated at 28°C for one month to generate colonies.