Antibiotics and antimycotic

Each disc of PCL or HAp-PCL was sterilized by a fully automated ethylene oxide gas sterilizer (SA-N160, Iki Co., Otsu, Japan). The rBMSCs were collected and cultured from the tibia and femur of 6-week-old male F344 rats according to the protocol in the previous article [49 (link)], and cells with the passage numbers of 3 and 4 were used for in vitro cell-based studies. The rBMSCs were cultured with α-MEM (FUJIFILM Wako Co., Osaka, Japan) containing 20% fetal bovine serum (FBS, Cytiva, Tokyo, Japan) and 1% antibiotics and antimycotics (Gibco Invitrogen, Carlsbad, CA, USA). The cells (3 × 10⁴ cells) were seeded onto each disc (PCL or HAp-PCL), and the discs were placed in a 24-well plate. The DNA concentration of cells 6, 12, and 24 h after seeding were measured using the Quant-iT™ PicoGreen™ dsDNA Assay Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA) according to the manufacturer’s instruction. After seeding rBMSCs on each disc (PCL or HAp-PCL) for 24 h, viability was assessed using the LIVE/DEAD Viability/Cytotoxicity Kit (Thermo Fisher scientific Inc., Waltham, MA, USA) according to the manufacturer’s instruction. The stained cells were observed by confocal laser fluorescence microscope (LSM-700, Zeiss Microscopy, Jena, Germany). Each experiment was performed in triplicate.

iNSCs were plated at 5000 cells per well on several 8-well chamber slides or 48-well plates, previously coated using recombinant human laminin. Then the iNSC were incubated neural differentiation media containing Neurobasal Media (Invitrogen) supplemented with N2 (1X, Invitrogen), B27 (1:50, Invitrogen), and 1% antibiotics and antimycotics (Invitrogen) for 21 days.

Spodoptera frugiperda 9 (Sf9) insect cells were propagated at 28°C in Sf-900 II medium (Invitrogen, Carlsbad, CA, USA) supplemented with antibiotics and antimycotics (Invitrogen). 293TT cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), hygromycin B (400 µg/ml; Invitrogen) and 1% penicillin/streptomycin (Gibco, Grand Island, NY, USA).

B6.129(Cg)-Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J mice were crossed to FVB.Cg-Tg(CAG-cre/Esr1)5Amc/J mice (Jackson Lab). E10.5 pregnant mice were intraperitonially injected with tamoxifen at dose of 1.2 mg per mouse. The sterna were collected from GFP-positive E12.5 embryos, the skin and most of the rib was removed. Sternums were cultured on Costar Transwell inserts (#3,450) ventral side down in DMEM/F12 (Gibco, Cat. #11,320) with 10% fetal bovine serum plus antibiotics and antimycotics (Gibco, Cat. # 15,240). Live imaging was performed using PerkinElmer spinning disc confocal microscopy at 37 °C and in 5% CO₂. Images were taken at time intervals of 5 min for up to 10 h. z-Stacks (1.5 or 2 μm) were generated using a × 20 objective lens with long working distance (3.5 mm) and water immersion. Both transmitted light and 488 nm laser were used to visualize sternums contour and individual GFP-positive sternum cells, respectively. Live images were analysed with Volocity software.

Human and rat derived basophils, KU812F and RBL-2H3 cells were cultured in RPMI-1640 and DMEM media containing 10% heat-inactivated FBS (Gibco BRL, USA), and antibiotics and antimycotics (Gibco BRL) under 37°C in a humidified atmosphere with 5% CO₂, respectively. Additionally, the cells were pretreated with 10, 25, 50, and 100 μg/ml A40 for indicated time and stimulated with 40 nM PMA (Sigma-Aldrich, USA) and 1 μM A23187 (Sigma-Aldrich) (PMACI) for KU812F cells and 10 μg/ml com 48/80 (Sigma-Aldrich) for RBL-2H3 cells under serum-free media.