B 27 plus

Duck neurons were isolated according to our previously described method for isolating mouse neurons (29 (link)). Briefly, the brain was collected from 9-day-old duck embryos, the meninges were peeled away and carefully removed, and the cortex was transferred into a new dish filled with HBSS, and then cut into 1-mm pieces by using scissors; the shears were transferred into 0.1% trypsin diluted in HBSS and digested for 20 min at RT. The trypsin was removed by transferring the brain cells into a new tube with proper DMEM, and then DNase I was added and treated for 5 min at RT. The DNase I was removed and the cells were collected by centrifugation at 1,200 rpm for 10 min at RT. Then, the collected cells were resuspended with DMEM and dissociated by repeated pipetting (<15 times). The cells were passed through a 75-nm nylon mesh (Corning, NY, USA) to separate the single cells, washed once in HBSS, and then cultured in D-polylysine–pretreated plates in DMEM supplemented with 5% FBS and 1% penicillin–streptomycin for 6 h. Finally, the cells were washed once with phosphate-buffered saline (PBS), and the medium was replaced with serum-free, neural-basal medium supplemented with 2% B-27 PLUS (Gibco-BRL, NY, USA), and the cells were incubated for 5 days to form a monolayer.

Primary neuron cells were isolated from the cerebral cortex of 16-day C57BL/6 mouse embryos as described previously [70 (link)]. Briefly, the cerebral cortex was separated from the brains and then dissociated with 0.25% trypsin (wt/vol) at 37°C for 30min. After centrifugation, the cells were resuspended in DMEM containing 5% FBS (Gibco) and subsequently passed through a 70 μm filter. After cell counting, the cells were seeded into 12-well plates (SORFA Life Science Co., Ltd, Beijing, China) that had been pretreated with poly-D-lysine and laminin (10 μg/ml) (Sigma, P7280). The cell supernatants were discarded at 5 h after seeding and replaced with Neurobasal plus Medium (Gibco, A3582901) containing 2% B-27 Plus (Gibco, A3582801). Then replace the medium every two days until 5–6 days post seeding for experiments.

Postnatal day 0–2 (P0–P2) mouse pups were sacrificed by decapitation. Cerebral cortices were dissected and minced prior to incubation in enzyme digestion solution (1 × hibernate complete medium, 0.06 mg/mL l-cysteine (Sigma, 778672), 1.4 × 10⁻² N NaOH (Sigma, 43617), 10 ng/mL APV (2-Amino-5-phosphonopentanoic acid, Sigma, A-5282), with 50 uL Papain (Worthington, LS 03126)) for 30 min at 37 °C. Following digestion, tissue was washed with DPBS and dissociated in complete Hibernate-A medium. Cells were seeded onto 0.1% PEI-coated glass coverslips at a density of 160,000 cells/cm². After 30 min, complete media change was performed with neurobasal complete medium (1 × Neurobasal Plus medium (Gibco, A3582901), 1% B27 Plus (Gibco, A3653401), 0.5 mM Glutamax Supplement (Gibco, 35050061), and 1 Penicillin/Streptomycin Solution (Gibco, SV30010)). Cells were incubated at 37 °C in 5% CO₂ for 14 days. Half-media changes were performed every 3–4 days with neurobasal complete medium.

Female, pregnant C57bl6 mice were
sedated and euthanized by cervical dislocation, and embryos were removed
from the uteri and decapitated. The embryo brains were exposed, and
cortices were removed. Cortices are transferred and triturated in
Hibernate E wo Ca²⁺ (A1247601, Gibco). The cells were collected
and seeded at densities of 20 000–50 000 cells/compartment
in Neurobasal plus (A3582901, Gibco), supplemented with 0.1% Gentamycin
(15710064, Gibco), 2% B27 Plus (A3582801, Gibco), 0.25% Glutamax (35050038,
Gibco), or NbActive (NbActiv4500, BrainBits LLC) with 0.1% Gentamycin.
All media was supplemented with 20 ng/mL NGF (556-NG-100/CF, R&D
System) and 20 ng/mL BDNF (450–02, Preprotech). A 50% media
exchange was performed once a week.

Removed brains were mounted and cut into coronal slices (300 μm) on a vibrating blade microtome (Leica) in cold HBSS supplemented with 100 U/ml penicillin/streptomycin, 10 mM HEPES, and 4.5 mM sodium bicarbonate. The SCN slices were dissected out and transferred onto a PTFE membrane insert (Millipore) in 35 mm culture dishes with 1.2 ml of DMEM (D5030, Sigma) supplemented with 3.5 g/L D-glucose, 2 mM Glutamax (Gibco), 10 mM HEPES, 25 U/ml penicillin/streptomycin, 2% B-27 Plus (Gibco), and 0.1 mM D-Luciferin sodium salt (Tocris). The SCN slice position was adjusted to the center of the dish and 1.5 μl AAV (pAAV1-Syn-ChrimsonR-tdT, Addgene) (Klapoetke et al., 2014 (link)) was placed directly onto the SCN slice. The culture dishes were then sealed with an optically clear PCR plate film (Bio-Rad) and maintained in a non-humidified incubator at 36.8 °C for about 10 days. The opsin expression was checked after about 10 days of viral transduction by imaging tdT fluorescence.