Gentamycin

Colons from naive or colitis mice were flushed 10 times with PBS to remove faeces. Two 5-mm-long segments of colon tissue were cultured in 250 μl of RPMI supplemented with 10% heat-inactivated foetal bovine serum, 1% penicillin–streptomycin and 50 mg/ml gentamycin (Wako) in a 48-well plate (Corning, NY) at 37 °C for 30 min [24 (link)]. OT in the culture supernatant was measured by an ELISA kit.

One day prior to the infection experiment, bacterial strains to be used were inoculated in selective LB broth overnight with constant agitation at 30°C. On the day of the experiment, bacterial cultures at the stationary phase were diluted 20-fold in serum-free DMEM (Sigma) and cultured with agitation at 37°C for 2 hours. Two hours before the actual infection, differentiated THP-1 was washed once with PBS and cell medium was changed to serum-free RPMI containing LPS at 1 μg/ml (Sigma). Upon completion of 2 hours of bacteria culture, bacteria were added to cell culture at m.o.i (multiplicity of infection) of 20 or as indicated and cells were subjected to 10 min of spin-infection at 1600 x rpm to synchronize the start of infection. Following 1 hr of incubation at 37°C, 5%CO₂, gentamycin (Wako) was added at concentration of 0.1 mg/ml to terminate further infection by extracellular bacteria. Cells were further incubated and samples (cells or cell culture medium) were harvested for analysis at time indicated.

As previously described [36 (link)], mice were treated with antibiotics, which were administered daily starting from seven days before the injection of α-synuclein PFF until the end of the experiments. The antibiotics were administered as a cocktail provided in sterile drinking water and included ampicillin (1 g/L; Nacalai Tesque, Kyoto, Japan), vancomycin (0.5 g/L; Shionogi Pharma), neomycin (0.5 g/L; Nacalai Tesque), gentamycin sulfate (100 mg/L; Nacalai Tesque) and erythromycin (10 mg/L; Sigma-Aldrich Japan, Tokyo, Japan). We prepared stock solutions of ampicillin, vancomycin and neomycin by dissolving them in sterilized water at a concentration of 50 mg/mL. erythromycin was dissolved in 99.5% ethanol (FUJIFILM Wako Pure Chemical) at a concentration of 50 mg/mL, while gentamycin was dissolved in sterilized water at concentration of 25 mg/mL. The stock solutions were aliquoted and stored at −30 °C for two months. For preparation of an antibiotic cocktail, the stock solutions were mixed with 300 mL of sterilized water. The average amount of water intake was 5 to 7 mL/mouse/day.

Primary neuronal cells were obtained from the hippocampus of 4-week-old wild-type and mutant mice (n = 5 mice/goup) as reported previously[13 (link)]. Briefly, the hippocampus was dissected and sliced into 0.5-mm sections in 2 mL HABG medium (40ml HA(Hibernate^TM-A Medium, Invitrogen, #A1247501; 0.8ml B27, Invitrogen, #17504; 0.1ml L-Glutamine, Invitrogen, #25030081)) at 4°C in a 35-mm-diameter dish using tissue slicer (Dosaka microslicer, Kyoto, Japan), removing the dentate gyrus to eliminate granule cells. The sections were digested with papain (2 mg/mL, Worthington, #LS003119 in HA-Ca, BrainBits LLC) at 30°C for 30 min. Cells were released by gentle trituration with a Pasteur pipette. Finally, primary neurons were separated using density-gradient centrifugation (OptiPrep, AXS, #1114542, XX). Cells were cultured in NeurobasalA/B27 medium (Invitrogen, #10888022 and #17504044) with L-Gin (Invitrogen, #25030149), growth factors (5 ng/mL mouse FGF2, Invitrogen, #PMG0034; 5 ng/mL mouse PDGF-BB, Invitrogen, #PMG0044), and gentamycin (Wako, #078–06061) for 1 week before the experiments.
To evaluate neuronal activity and mitochondrial quantity, cells were fixed with 4% paraformaldehyde for 20 min and then processed for the detection of neuronal antigens. The primary antibody was MAP2 (ab5392, Abcam) at 1:2000.