25 cm2 t flask

Mouse microglial cells (N9) were kindly provided by Dr. Bai Yun (Department of Genetics, The Third Military Medical University, China). The N9 microglia was obtained by immortalization of E13 mouse embryonic brain cultures with the 3RV retrovirus carrying an activated v-myc oncogene [33] (link) and shares many phenotypical characteristics with primary mouse microglia [34] (link). N9 cells were maintained in Iscove's modified Dulbecco's medium (IMDM) (HyClone, Logan, UT) supplemented with 5% heat-inactivated fetal bovine serum (HyClone), 2 mM glutamine, 100 µg/mL streptomycin, 100 U/mL penicillin (Beyotime, Jiangsu, China) and 50 µM 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA). Mouse astrocyte type I cells (C8-D1A) (ATCC, Rockville, MD, USA) were cultured in Dulbecco's Modified Eagle's medium (DMEM) (GIBCO, Gran Island, NY, USA). Culture medium was supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 µg/mL streptomycin (Beyotime). Both microglial and astroglial cells were plated in 25-cm² T-flasks (Corning, Tewksbury, MA, USA) and incubated at 37°C in a humid atmosphere with 5% CO₂.

Spodoptera frugiperda 9 (Sf9) cell line was plated in 25-cm² T-flasks (Corning) and maintained with TC-100 medium (Gibco) containing 10% heat-inactivated fetal bovine serum (FBS; Gibco) and streptomycin-penicillin solution (100 IU/mL penicillin and 100 mg/mL streptomycin, Amresco) at 27°C.
For transfection, cells were seeded in 96-well cell culture plates and transfected with pGL4-Vg Luc reporter and pRL-TK expression vector (the ratio of the two vectors (w/w) was 10: 1) using X-treme GENE HP DNA Transfection Reagent (Roche, USA) following the manufacturer’s instructions. For cotransfection of the BlBrC-Z protein (1–4), the ratio (w/w) of the reporter vector with the BlVg promoter, pBmFlag-BlBRC-Z (1–4) and the pRL-TK expression vector was 10:10:1. At 6 h after transfection, the culture medium was replaced with fresh medium containing FBS. After 24 h of transfection, the cells were treated with 0.2 μg/ml 20-hydroxyecdysone (20E) (Sigma, USA) or JH-III, which were dissolved in dimethyl sulfoxide (DMSO) at a stock concentration of 1 mg/ml and stored at -20°C. Controls were treated with 0.1% DMSO only. Cells were cultured for an additional 24 h and then lysed to detect luciferase activity with the Dual-Luciferase Reporter Assay Kit (Promega, USA) using a GloMax 20/20 Luminometer (Promega, USA) according to the manufacturer’s instructions. All assays were performed in triplicate.

The brain tissue of neonatal offspring SD rats (after birth within 24 h) was rinsed with phosphate-buffered saline (PBS), and then the hippocampus and striatum tissues were isolated and mechanically cut into 1 mm³ sections with ophthalmic surgical scissors and transferred to serum-free medium Dulbecco’s modified Eagle’s medium (DMEM) and nutrient mixture F-12 Ham (F12) (1:1) (Corning, New York, NY, USA) with 2% B27 (Gibco, Billings, MT, USA), 20 ng/mL epidermal growth factor (EGF; PeproTech, Cranbury, NJ, USA), 20 ng/mL basic fibroblast growth factor (bFGF; PeproTech), and 2 mmol/L L-glutamine (Sigma, St. Louis, MO, USA). Single-cell suspensions were isolated with a straw blower, seeded at 1 × 10⁶/cell/mL in 25-cm² T-flasks (Corning), and cultured at 37 °C in a 5% CO₂ atmosphere for 7 days. Purified NSCs were cultured in a medium containing different concentrations of folic acid for 7 days (D-D group and D-N group, 0 μM; N-D group and N-N group, 10 μM). After 10 days of culture under different conditions, the cells were harvested to detect proliferation and apoptosis ability, as well as telomere attrition.

The cell lines used were the human colorectal cancer cell lines HT-29, SW-480 and HCT-116 (Sigma-Aldrich). Cells were cultured in a high glucose (4.5 g/L) medium, Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin mixture. During the experiment, cells were kept in 25-cm² T-flasks (Corning, NY, USA) under the standard conditions of 37 °C and 5% CO₂.

N. fowleri used in this study was a reference CDC VO 3081 strain from Dr. GS Visvesvara at the Centers for Disease Control and Prevention (USCDC). The amoeba was axenically cultured in Nelson’s medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) (GE Healthcare Lifesciences, Logan, UT, USA) at 37 °C in T 25-cm² flask (Corning, New York, NY, USA) in the secure facility of the Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok.
Three isolates of unidentified environmental bacteria, designated KP-01, KP-14, and KP-15, were randomly selected from a culture stock of bacteria isolated from a fresh water canal in Bangkok. These bacteria were grown in Luria–Bertani (LB) broth at 37 °C with shaking aeration until the stationary phase of growth.

N. fowleri was grown in Nelson’s medium supplemented with 10% FBS at 37 °C in a T 25 cm² flask (Corning, New York, NY, USA) in a secure facility.