Anaero pouch anaero

ASCs were cultured under normoxic conditions in complete DMEM up to 80% confluency for all experiments. Two different methods were used to induce hypoxia at 2% and <0.1% O₂ concentration. For the 2% O₂ concentration, ASCs were cultured in a humidified incubator (Thermo) at 37°C, 2% O₂. For a hypoxic environment with less than 0.1% O₂, the hypoxia system Anaero Pouch–Anaero; (Mitsubishi Gas Chemical Company Inc., Japan) was used as previously described (Stubbs et al., 2012 (link)). The final indoor gas compositions were <0.1% O₂ and 15% CO₂. In order to mimic ischemia and anoxia in vitro, the OGD model was established using a previously published method (Chen et al., 2019 (link)). Similarly, in our study, the OGD exposure means that ASCs were cultured in serum- and glucose-free DMEM under hypoxic conditions (<0.1% O₂) for 24 h.
To evaluate the effect of the degree and duration of hypoxia on ASCs exposed to OGD, the following experimental groups were established:

Lactiplantibacillus plantarum OLL2712 was isolated in our laboratory and deposited in the International Patent Organism Depositary (Chiba, Japan) under accession No. FERM BP-11262, which was used in this study. The other bacterial strains are listed in Table 1. They were cultured in de Man–Rogosa–Sharpe broth (MRS; Becton Dickinson, Franklin Lakes, NJ, USA) at 37 °C for 18 h under anaerobic conditions with AnaeroPouch-Anaero (Mitsubishi Gas Chemical, Tokyo, Japan). The bacterial cells were harvested and washed twice with phosphate-buffered saline (PBS; pH 7.2), then washed once with distilled water. The cells were then heat-treated at 75 °C for 60 min and then freeze-dried. The lyophilized cells were resuspended in distilled water at a concentration of 10 mg/mL and used for in vitro assays.

A spoonful (0.5–1.0 g) of the first intestinal discharge (obtained from the first diaper) was collected fresh in a fecal collection tube (Sarstedt AG & Co., Numbrecht, Germany) containing 2 ml RNAlater (Ambion, Austin, TX, USA). Out of total 151 samples, 148 were passed within 24 h after birth whereas the rest three samples were discharged between 24 and 48 h. As described elsewhere (Tsuji et al., 2012 (link)), stool samples were also collected at age 3 and 7 days, 1, 3, and 6 months, and 3 years for follow-up analyses. All samples were collected at the hospital by following routine and standard clean techniques such as the use of sterile sample collection tube and spatula, and handler’s mouth masked, hands sanitized and gloved, and head capped while retrieving the sample. Immediately after collection, samples were stored in the refrigerator (3–4°C) anaerobically by using Anaero Pouch-Anaero (Mitsubishi Gas Chemical Company, Inc., Tokyo, Japan) and were sent immediately in a cooling box with refrigerants and anaero-packs to the research lab where these were stored at 3–4°C in a Biosafety Category II microbiology laboratory until further processing.