Anaeropack kenki

Blautia coccoides was inoculated in yeast-peptone-dextrose (Becton, Dickinson and Company, NJ, USA) medium containing sodium cholate (0.0015 % v/v) and glucosylceramide (4 μg/μL in ethanol) or vector ethanol (1 % v/v) at a cell density of 1 × 10⁶ cells/mL. The bacterial cells were incubated in anaerobic jars (AnaeroPack Kenki, Mitsubishi Gas Chemical Co., Inc., Tokyo, Japan) at 30 °C for 24 h. Cultures were homogenized and the OD₆₀₀ was measured using a spectrophotometer (UV-1800; Shimadzu, Kyoto, Japan). To incubate Lactobacillus casei, MRS medium (Becton, Dickinson and Company) was used; to incubate Escherichia coli, Luria-Bertani (LB) medium (Nissui, Tokyo, Japan) was used.

The strain of E. faecalis purchased from NBRC (NBRC No. 100481) was used in this study. E. faecalis was restored by 310 medium and cultured anaerobically at 37 °C using an anaerobic jar (Mitsubishi gas chemical, Tokyo, Japan) and AnaeroPack^® Kenki (Mitsubishi gas chemical, Tokyo, Japan). The culture was stored at −80 °C in 15% glycerol.

Brain-Heat Infusion (BHI) agar was purchased from Becton Dickinson and Company (Franklin Lakes, NJ). Modified GAM broth and modified GAM agar were purchased from Nissui Pharmaceutical Co., LTD (Tokyo, Japan). Porphyromonas gingivalis ATCC 33277, Fusobacterium nucleatum ATCC 25586, Streptococcus sanguinis ATCC 10556, and Streptococcus mutans ATCC 25175 were purchased from the American Type Culture Collection (Manassas, VA). P. gingivalis and F. nucleatum were grown on GAM broth and GAM agar, and S. sanguinis and S. mutans were grown on BHI broth and BHI agar in an anaerobic chamber with Anaeropack Kenki (Mitsubishi Gas Chemical Company, Tokyo, Japan) at 37 °C.

Feces from the AIMD (n = 13) and control (n = 12) mice were collected at ZT 0–2 and immediately stored under anaerobic conditions (AnaeroPack-Kenki; Mitsubishi Gas Chemical Co., Ltd., Tokyo, Japan). Here, 20 mg of fecal grains was diluted in 1 mL of PBS, and this solution was diluted 10³-, 10⁵-, and 10⁷-times with PBS before culturing on Gifu anaerobic medium (GAM) agar for 24 h under anaerobic conditions at 37 °C. The number of colonies was counted following 24 h of culture to estimate the number of viable bacteria per gram.

After the aggregates were transferred to adhesion plates, the culture medium was changed to Neurobasal Medium (NM, Gibco, Life Technologies Corporation, Grand Island, NY, USA), containing 0.5 mM of L-Glutamine (Gibco) and 2% NeuroBlew-21 (MACS) on D26. Q-VD-Oph (10 µM; Abcam, Cambridge, UK), Necrox-5 (30 µM; Enzo, Farmingdale, NY, USA), and Z-LEHD-FMK (10 µM; Medical & Biological Laboratories, Tokyo, Japan) were added to the respective plates on D26. On D27, the plates were transferred and sealed in a container with AnaeroPack Kenki (Mitsubishi Gas Chemical, Tokyo, Japan), an oxygen absorber/carbon dioxide generator, to simulate hypoxic injury at 37 °C for 18 h. The plates were then removed from the container and incubated at 37 °C for 4 h under normal atmospheric conditions to simulate reoxygenation. The duration of hypoxia and reperfusion was deemed appropriate based on a previous report [19 (link)], and we further conducted multiple experiments in order to adjust the conditions under which both cell death and their rescue by the drug could be observed.

Fresh fecal samples were collected immediately after defecation avoiding contamination from the environment and kept under anaerobic conditions with AnaeroPack^® Kenki (Mitsubishi Gas Chemical Company Inc., Tokyo, Japan). In the case of the pre-weanling group, kittens were provoked to defecate. Samples were refrigerated and transported to Laboratory of Veterinary Public Health, the University of Tokyo, the next day. Samples from the senile cats were aseptically collected immediately after defecation at their veterinary clinic and transferred to the laboratory in the same way.