Cell culture dishes

Mice 6–10 weeks of age were euthanized with carbon dioxide (CO₂) followed by cervical dislocation and sterilization of femurs and tibias in 70% ethanol. To obtain the mouse BMDM, proximal and distal ends of the femurs and tibias were transversally cut and the bone marrow was flushed out by injecting 5 ml of Corning Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated FBS (Fisher Scientific), 5% pen-strep (Life Technologies), and 5% l-glutamine (Life Technologies) into the medullary canal with a syringe and 25-gauge needle. Cells were centrifuged and resuspended in DMEM and further supplemented with 10% L929 cell-conditioned medium. Cells were plated into four 10 cm diameter cell culture dishes (BD, Franklin Lakes, NJ) and cultured for 5 days at 37 °C and 5% CO₂. After reaching confluence, cells were lifted with gentle scraping, centrifuged, and split into six-well culture dishes and stimulated with the TLR4 ligand LPS (Invivogen, Cat. # TLRL-3pelps).

General reagents were purchased from Nacalai Tesque (Kyoto, Japan). Gold(III) chloride, sodium borohydride, PSS, PDDAC, PEI, and PLL were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Silver nitride, L(+)-ascorbic acid, sodium oleate, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), sodium cholate, Gold ICP-MS Standard, and the lactate dehydrogenase (LDH) assay kit were obtained from Wako (Osaka, Japan). Rho-PE and DOTAP were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). Opti-MEM, LF2000, LysoTracker Green DND-26 (LysoTracker), and fetal bovine serum (FBS) were obtained from Life Technologies (Carlsbad, CA, USA). Escherichia coli strain BL21 was purchased from Novagen (Madison, WI, USA). Spectra/Por Dialysis membranes (MWCO 50 kDa) were purchased from Spectrum Laboratories (Rancho Dominguez, CA, USA). NAP-5 columns were purchased from GE Healthcare UK Ltd. (Buckinghamshire, UK). Cell culture dishes and trypsin/EDTA were obtained from BD Biosciences (San Jose, CA, USA). Glass-based dishes with or without grid lines were purchased from Matsunami Glass Co. Ltd. (Osaka, Japan). Annexin V-FITC and propidium iodide were obtained from Funakoshi (Tokyo, Japan). Cell Counting Kit-8 was obtained from Dojindo (Kumamoto, Japan).

Human periodontal ligament stem cells were isolated and cultured according to a previously reported protocol with minor modifications [2 (link)]. In brief, human premolars (n = 20) were extracted for orthodontic reasons from 10 healthy adult volunteers. Periodontal ligament tissues were separated from the root surface using a scalpel and were minced into the smallest size possible. The minced periodontal ligament tissues were digested for 45 min in digestive enzymes (3 mg/mL collagenase type I [Sigma, USA] and 4 mg/mL Dispase II [Sigma, USA]) at 37°C. Single-cell suspensions were seeded onto cell culture dishes (BD, USA) containing growth medium of a-MEM (Hyclone, USA) supplemented with 20% fetal bovine serum (FBS; Gibco, USA) and then incubated at 37°C with 5% CO₂. Single-cell colonies were observed and passage 0 (P0) cells were cultured. Then, passages 3 through 5 (P3–P5) cells were used for this study.

The p50 strain of the silkworm, Bombyx mori, was grown on the fresh leaves of the mulberry, Morus bombycis. The female larvae of the fifth instar were aseptically dissected 3 days after the fourth ecdysis and the fat body was isolated. More than 100 chunks of tissue (approximately 2 mm³) were excised from the fat bodies of 108 larvae. Those tissue particles were incubated in cell culture dishes (diameter, 35 mm; BD Biosciences, Franklin Lakes, NJ, USA) with MGM-450 insect medium [17 ] supplemented by 10% fetal bovine serum (BioWest, Nuaillé, France) with no gas change. The tissue was cultured for 80 hours at 25°C. The microbes were checked for infection using a microscope. Infection-free tissues were used in the following induction assays. No antibiotics were used in the assays to maintain the primary culture.

Cells were stimulated overnight with 10, 100 or 250 nM of OT (Bachem, Bubendorf, Switzerland) in cell culture dishes or chamber slides (BD Falcon, Germany). For the inhibition of CaN, CaN autoinhibitory peptide (#1891, Tocris, Wiesbaden, Germany) was used at a concentration of 10 µM [68 (link)]. Cells were pretreated with the inhibitor for 30 min and stimulated with the corresponding treatment. Non-treated cells are indicated as vehicle (VEH).