Thermomixer c shaker

Stool samples were adjusted to 0.2 g/mL with sterile PBS followed by homogenization for 20 min at 1400 rpm and 4 °C using a Thermo Mixer C shaker (Eppendorf, Hamburg, Germany). The nose and cecum were weighed, transferred to autoclaved homogenizer tubes containing zirconium oxide beads (diameter: 1.4/2.8 mm, Precellys, France), and filled up with 1 mL sterile PBS. Cecum samples were homogenized at 6000 rpm for 2 × 20 s with a 15 s interval. Noses were homogenized at 6500 rpm for 2 × 30 s with a 5 min interval. Homogenized samples were serially diluted; 10 µL of each dilution were plated out in triplicate on S. aureus Chromagar plates (CHROmagar, France) and enumerated the next day.

Assessment of TRITC-BSA and FM4-64 absorption efficiency was performed as previously described (Watterson et al., 2022 (link)). In brief, approximately 100 day 5 (hsf-1 RNAi), day 3 (rfip-2 RNAi), or day 1 (act-5 RNAi) adult worms were washed three times in M9 buffer and then incubated in 50 μL of 15 mM TRITC-BSA (Thermo Fisher Scientific A23016) or 0.4 mM FM4-64 (Thermo Fisher Scientific T3166) in an Eppendorf Thermomixer C shaker set to 300 rpm for 2 h at 20°C. Worms were then washed three times with M9 buffer and allowed to recover on NGM plates seeded with OP50 for 2 h in the dark. For quantification of endocytosis efficiency, worms were visualized under an Axio Observer inverted microscope (Carl Zeiss Microscopy) at 50x magnification and scored according to absorption efficiency; absorption was considered impaired if less than 50% of TRITC-BSA/FM4-64 was absorbed across the intestinal epithelia. For imaging, worms were rinsed off plates with M9, centrifuged (1,000 x g) for 30 s, and mounted on slides in M9 supplemented with 100 mM levamisole.

Female mosquitoes were collected 5–7 days post-eclosion and weighed in groups of 25 in 1.5 mL microcentrifuge tubes. The weight of single mosquitoes was estimated from these measurements to reduce error. To obtain acid resistant material, 25 female mosquitoes were homogenized in 500 μL diH₂O using an electric pellet pestle cordless motor (Kimble). After homogenization, 500 μL of 12 M HCl was added to the homogenate (6 M final concentration). Samples were digested for 24 h on an Eppendorf ThermoMixer C shaker at 85 °C with 700 RPM shaking. After digestion, samples were spun down for 30 min at room temperature at 15,000 RCF. Samples were washed three times, first with 1 mL 1× PBS, then 1 mL 10% PBS, and finally 1 mL diH₂O. Washed melanin samples were lyophilized, weighed, and combined for ssNMR analysis.