Hanks balanced salt solution (hbss)

Primary cortical neurons were isolated from mice embryos at embryonic day 14; details of the isolation method have been described in a previous report.²¹ (link) Cerebral cortex tissue samples were dissected, removed, and immediately placed into 500 μl of ice-cold Hank's Balanced Salts (HBSS) (Sigma). The supernatant of HBSS was removed using a small desktop centrifuge (Millipore). 0.25% trypsin-EDTA was added to incubate the samples at 37 °C for 10 min. Following removal of trypsin-EDTA, the tissue samples were treated with DNase (final 50 mg/ml) that was incorporated into the HBSS. After employing centrifugation to remove the DNase in the HBSS, 500 μl of HBSS containing 20% (v/v) fetal bovine serum (FBS) was added. A pipette was used to dissociate the tissue pellets from the HBSS containing 20% FBS, and the dissociated samples were then centrifuged at 400 rcf for 5 min using a centrifuge (Centrifuge 5418, Eppendorf). This process was repeated until the cells had been sufficiently dissociated. All animal experimental procedures were approved by The Institutional Animal Care and Use Committee at Tokai University.

The OU isolation process was adapted from our previously published protocol (Sala et al., 2011 (link); Grant et al., 2015 (link); Hou et al., 2018 (link)). Two-week-old C57BL/6 mice were euthanized in a CO₂ chamber and the small intestine was collected and opened longitudinally. The resected tissue was vigorously washed five times with cold Hank's Balanced Salt Solution (HBSS, 14170-112, Life Technology) containing Antibiotic/Antimycotic (Anti/Anti, 15240062, Life Technology) to remove the intestinal content. The intestines were minced into 1 × 1 mm² pieces, washed twice in cold HBSS/Anti/Anti, sedimented at 164 g (Eppendorf 5810R−1,000 rpm) between washes, and digested with type IV collagenase (800 units/ml, CLS-4, Worthington) and dispase (0.12 mg/ml, 17105-041, Gibco) in HBSS for 20 min at 37°C. The digestion was stopped with 10% fetal bovine serum (FBS, Invitrogen) in high-glucose Dulbecco's modified Eagle's medium (DMEM, 10566-016, Invitrogen), the sediment (pellet) was minced with a pipette and centrifuged at 41 g (Eppendorf 5810R−500 rpm) for 5 min to remove single cells. The resulting pellet containing the OU was resuspended in DMEM supplemented with 10% FBS, non-essential amino acids (NEAA, 11140-076, Life Technology) and Anti/Anti, centrifuged one last time at 105 g (Eppendorf 5810R−800 rpm) and the supernatant was removed.