Penicillin streptomycin mixed solution

Human lung adenocarcinoma A549 cells (JCRB0076) and human fibrosarcoma HT-1080 cells (JCRB9113) were provided by the National Institutes of Biomedical Innovation, Health and Nutrition JCRB Cell Bank (Osaka, Japan). A549 cells, HT-1080 cells, and human breast carcinoma MCF-7 cells were maintained in RPMI 1640 medium (Thermo Fisher Scientific, Grand Island, NY, USA) supplemented with heat-inactivated fetal calf serum (Nichirei Biosciences, Tokyo, Japan) and penicillin-streptomycin mixed solution (Nacalai Tesque, Kyoto, Japan).

The epididymides were surgically excised at a region of the vas deferens close to the tail of epididymis, and those with testis were dissected from the embryonic body. Samples were then placed on a hydrophilic polytetrafluoroethylene organ culture insert with a pore size of 0.4 μm (Merck Millipore, PICM01250), which was preset in a 35 mm Petri dish (Thermo Fisher Scientific, 153066) filled with a culture medium. The culture medium we used was Dulbecco's Modified Eagle's medium (Nacalai Tesque, 08489-45), containing 10% fetal bovine serum (Thermo Fisher Scientific, 12483) and 1% penicillin-streptomycin mixed solution (Nacalai Tesque, 26253-84) at 37°C under 5% CO₂. The samples were cultured in an air-liquid interface; the total amount of culture medium was 800 μl for the Petri dish.

293T embryonic kidney cells (#RCB2202; Riken BioResource Research Center, Tsukuba, Japan) and G355‐5 feline astrocyte cells (#CRL‐2033; American Type Culture Collection, Manassas, VA, USA) were added to Dulbecco's Modified Eagle's Medium (#5796; Sigma‐Aldrich, Tokyo, Japan) with inactivated fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) and Penicillin–Streptomycin Mixed Solution (#09367‐34; Nacalai Tesque, Kyoto, Japan) and cultured at 37 °C and 5% CO₂ levels.

HeLa (#CCL-2.2, ATCC), FLAG-RIG-I/HeLa (derived from HeLa; #CCL-2.2, ATCC), FLAG-IPS-1/HeLa (derived from HeLa; #CCL-2.2, ATCC) [33 (link)], EGFP-G3BP1/HeLa (derived from HeLa; #CCL-2.2, ATCC) [35 (link)], HEp-2 (#CCL-23, ATCC), BHK21 (#CCL-10, ATCC) cells, and MEFs (isolated from embryos under C57BL/6 background, Japan SLC, Inc.) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) (Nacalai Tesque) supplemented with 10% Fetal Bovine Serum (FBS) (BioWest) and 1% Penicillin-Streptomycin Mixed Solution (100 U/ml and 100 μg/ml respectively) (Nacalai Tesque).

Female 6‐week‐old BALB/c mice (CLEA Japan Inc., Tokyo, Japan) were used in the study. All mice were maintained in a temperature‐controlled, pathogen‐free room and were handled according to the approved protocols and guidelines of the Animal Committee of Osaka University (Suita, Japan). All animal experiments performed were approved by The Institute of Experimental Animal Sciences, Faculty of Medicine, Osaka University (Approval number: J007418‐004). NS‐1 and 4T1 mammary carcinoma cell lines were maintained in Roswell Park Memorial Institute 1640 (RPMI1640) medium (Nacalai Tesque Inc., Kyoto, Japan), and MC38 colon adenocarcinoma and B16‐F10 melanoma cell lines were maintained in Dulbecco's Modified Eagle Medium (DMEM) (Nacalai Tesque Inc.). Both RPMI1640 and DMEM complete media were supplemented with 10% fetal bovine serum (BioWest, Nuaille, France) and 0.1 mg·mL⁻¹ penicillin–streptomycin mixed solution (Nacalai Tesque Inc.). All cell lines and hybrid cells were cultured at 37 °C in a humidified atmosphere of 5% CO₂. Experimental animal sacrifice by carbon dioxide animal euthanasia.

Male C57BL6/J mice (10-weeks-old) were used to prepare peritoneal macrophages and bone marrow derived macrophages (BMDMs). The peritoneal macrophages were obtained by flushing the peritoneal cavity two times with 5 mL of ice-cold culture medium containing RPMI-1640 (Nacalai Tesque, Kyoto, Japan), 10% FBS, 0.1% 2-mercaptoethanol (Thermo Fisher Scientific, Waltham, MA, USA), and 1% penicillin-streptomycin mixed solution (Nacalai Tesque) using a 25-gauge needle. The peritoneal fluid collected was poured into a 100 mm dish, followed by incubation for 4 hr in a CO₂ incubator to isolate the cells attached to the bottom of the dish. After incubation, the dish was washed two times with 10 mL of pre-warmed culture medium, with the attached cells then collected by gentle scraping into 10 mL of culture medium for use in the experiments as peritoneal macrophages. For preparation of BMDMs, bone marrow cells were collected from femurs and tibias by flushing these bones with 10 mL of culture medium. After lysis of the red blood cells with 2 mL of hypotonic ammonium chloride solution, the cells were collected by centrifugation at 500 x g for 5 min at 4°C, and then cultivated for 7 d in 10 mL of culture medium containing recombinant mouse M-CSF (Pepro Tech, Cranbury, NJ, USA) at a concentration of 10 ng/mL.

Mouse neuroblastoma N2a cells (Okado-Matsumoto and Fridovich, 2002 (link)) were cultured in Dulbecco's Modified Eagle's Medium (DMEM high-glucose, Nacalai Tesque, Japan) in a humidified cell incubator at 37°C in a 5% CO₂ atmosphere. The medium was supplemented with 10% (v/v) fetal bovine serum (FBS; Biosera, France) and 1% (v/v) Penicillin-Streptomycin Mixed Solution (Nacalai Tesque).
Mitochondrial translation was inhibited in N2a cells by treatment with chloramphenicol (Nacalai Tesque) (Sun et al., 2016 (link)). 2×10⁶ cells were cultured in tissue culture dishes (100×20 mm, Thermo Fisher Scientific). After 18 h of cell culture, 40 μg/ml chloramphenicol was added into the culture medium, and the cells were incubated for 120 h. The chloramphenicol-treated culture medium was changed every 24 h. Then the chloramphenicol-treated cells were harvested and washed three times with phosphate-buffered saline, and stored at −80°C until use.