The virus strain DV3P12/08 was derived from patients infected with DENV-3 in Thailand in 2008, who exhibited DHF grade 1. The virus was isolated in Aedes albopictus C6/36 cells and then passaged 3–5 times in C6/36 cells and once in Vero cells. Virus stocks were stored at -80°C until use. The C6/36 cell line was cultured at 28°C in Leibovitz’s 15 medium (Gibco, Gland Island, NY) supplemented with 0.3% BactoTM Tryptose Phosphate Broth (Becton Dickinson, Sparks Glencoe, MD, USA) and 10% fetal calf serum (FCS). Vero cells were cultured in Eagle’s minimum essential medium (Nacalai Tesque, Kyoto, Japan) supplemented with 10% FCS.
Leibovitz s 15 medium
Manufactured by Thermo Fisher Scientific
Sourced in United States
Leibovitz's 15 medium is a cell culture medium formulated by Leibovitz for the growth and maintenance of a variety of cell types. It is a chemically defined medium that provides essential nutrients and growth factors required for cell proliferation and survival.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Mic 101 modular incubator, by Embrient Inc (2 mentions)
Eagle s minimum essential medium, by Nacalai Tesque (1 mentions)
Albumax 2, by Thermo Fisher Scientific (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Insulin transferrin selenium, by Merck Group (1 mentions)
6 well culture plate, by Corning (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Dmem medium, by Thermo Fisher Scientific (1 mentions)
Cell counting kit, by Vazyme (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Sw620, by American Type Culture Collection (1 mentions)
Dld 1, by American Type Culture Collection (1 mentions)
Sw620, by Thermo Fisher Scientific (1 mentions)
Ts100 inverted microscope, by Nikon (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Glutamine, by Merck Group (1 mentions)
9 protocols using leibovitz s 15 medium
1
Dengue Virus Strain Propagation
2
Ovarian Organ Culture Modeling
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Ovaries were dissected from PD4 mice and washed three times in Leibovitz’s-15 medium (Gibco) containing 10% fetal bovine serum plus 1% penicillin–streptomycin before being transferred to culture inserts (Millipore) in a 6-well culture plate (Costar) at 37℃ and 5% CO2. DMEM/F12 (Gibco) supplemented with 5% Insulin-Transferrin-Selenium (Sigma), 1 mg/ml BSA (Sigma), 1 mg/ml Albumax II (Gibco), 100 µM L-ascorbic (Sigma), and 1% penicillin–streptomycin was used as the culture medium. A drop of medium was placed to cover the top of the ovary to prevent drying, and the ovaries were cultured for 4 or 8 days with medium changed every 2 days. Ovaries were treated with control medium (1% DMSO), VCD (30 µM), VCD + MSC-CM (fivefold concentration), or VCD + Fib-CM (fivefold concentration). Appropriate concentrations of recombinant human HGF (100–800 ng/ml), G-CSF (100–800 ng/ml), BDNF (100–800 ng/ml), or HGF neutralizing antibody (0–1 ng/ml) were added directly to the culture medium.
3
Establishing Breast Cancer Cell Lines
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Both human and mouse breast cancer cell lines were obtained from Renji Hospital, Shanghai Jiaotong University, School of Medicine, including MCF-7, T-47D, MDA-MB-231, BT-20, and 4T1. All the cells except MDA-MB-231 and T-47D were maintained in DMEM medium (Gibco, USA), supplemented with 10% Fetal Bovine Serum (Gibco, USA) and 1% Penicillin–Streptomycin (Gibco, USA). MDA-MB-231 was maintained in Leibovitz's 15 medium (Gibco, USA) while T-47D was maintained in RPMI 1640 (Gibco, USA), both supplemented with 10% Fetal Bovine Serum (Gibco, USA) and 1% Penicillin–Streptomycin (Gibco, USA). Cells were cultured at 37 ℃ with 5% CO2.
4
Metabolite Modulation of HIRRV Infection in Flounder
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To evaluate the effect of differentially expressed metabolites on HIRRV-infected flounder, specific metabolites, including inosine, carnosine and glutamine, glycine, and 7-methylguanine, were selected for experimental verification after exposure to HINAE cells after virus infection. Specifically, HINAE cells were cultured in 6-well plates at 20°C in Leibovitz’s -15 medium (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA), 100 IU/mL penicillin and 100 μg/mL streptomycin. For experimental group, the cells were infected with HIRRV at a multiplicity of infection (MOI) of one for 1 hour. Subsequently, inosine, carnosine glutamine, glycine, 7-methylguanine were diluted with PBS, and added to HINAE cell at a final concentration of 0.15 mM, 0.1 mM, 4 mM, 6.8 mM, and 0.1 mM, respectively. After 48 h, total RNA of cells was extracted to detect copy number of HIRRV by RT-qPCR. The cytotoxicity of these metabolites were determined by Cell Counting Kit (CCK-8,Vazyme) 24 hours after supplement.
In addition, to validate our metabolomics results, we also measured the expression of 5’-methylthioadenosine (MTA), GSH, and cystine metabolism-related genes in the infected group of the late metabolite group. All experiments were performed in triplicate, and primers used in this study was shown inTable S1
In addition, to validate our metabolomics results, we also measured the expression of 5’-methylthioadenosine (MTA), GSH, and cystine metabolism-related genes in the infected group of the late metabolite group. All experiments were performed in triplicate, and primers used in this study was shown in
5
Culturing Colon Cancer Cell Lines
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The human colon cancer cell line DLD-1 and the human colon cancer lymph node metastasis cell line SW620 were purchased from the American Type Culture Collection (USA). The DLD-1 cell line was cultured in RPMI 1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The SW620 cell line was cultured in Leibovitz’s 15 medium (Gibco) supplemented with 10% FBS (Thermo Fisher Scientific, Inc.).
6
Hypoxia Response of LYCF Cell Line
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The LYCF cell line was kindly provided by Dr. You-Hua Huang (South China Agricultural University, Guangzhou). The LYCF cells were cultured in Leibovitz’s-15 Medium (Gibco, USA) supplemented with 10% (v/v) fetal bovine serum (Gibco, USA) and 200 µg/mL penicillin-streptomycin (Gibco) at 27 °C. For the hypoxic challenge, LYCF cells were cultured in a MIC-101 modular incubator (Billups Rothenberg Inc., USA) with 1% O2 and 99% N2 for 0, 3, 6, 12, 24, and 48 h.
7
Live-cell Imaging of HaloTag-expressing Cells
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Cells were seeded on ZMW coverslips
in a six-well plate at 40% to 45% confluency 1 day before the
imaging experiment. The cell culture medium was replaced with imaging
medium (prewarmed CO2-independent Leibovitz’s-15
medium (Gibco) with 5% fetal bovine serum and 1% penicillin/streptomycin)
30 min prior to imaging. All live-cell imaging experiments
were performed at 37 °C. For experiments with HaloTag-expressing
cell lines, the cell culture medium was replaced with the imaging
medium (prewarmed Leibovitz’s-15 medium with 5% fetal bovine
serum and 1% penicillin/streptomycin) containing 5 nM JFX650-Halo
ligands. After 10 min of incubation with the Halo ligands,
the cells were washed three times with fresh imaging medium.
in a six-well plate at 40% to 45% confluency 1 day before the
imaging experiment. The cell culture medium was replaced with imaging
medium (prewarmed CO2-independent Leibovitz’s-15
medium (Gibco) with 5% fetal bovine serum and 1% penicillin/streptomycin)
30 min prior to imaging. All live-cell imaging experiments
were performed at 37 °C. For experiments with HaloTag-expressing
cell lines, the cell culture medium was replaced with the imaging
medium (prewarmed Leibovitz’s-15 medium with 5% fetal bovine
serum and 1% penicillin/streptomycin) containing 5 nM JFX650-Halo
ligands. After 10 min of incubation with the Halo ligands,
the cells were washed three times with fresh imaging medium.
8
Hypoxia and ROS Modulation in LYCF Cell Line
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The large yellow croaker fry (LYCF) cell line was kindly provided by Dr. You-Hua Huang, South China Agricultural University. The LYCF cells were cultured in Leibovitz’s-15 Medium (Gibco, USA) supplemented with 10% (v/v) fetal bovine serum (Gibco, USA) and 200 µg/mL of penicillin-streptomycin (Gibco, USA) at 27 °C. For the normoxic conditions, the cells were cultured in an incubator. For the hypoxic treatments, the cells were cultured under hypoxic conditions at 1% O2 and 99% N2 in a MIC-101 modular incubator (Billups Rothenberg, USA) for 0, 1, 3, 6, 12, 24, and 48 h. For the ROS modification experiments, the functions and concentrations of ROS scavengers (N-acetylcysteine (NAC) and elamipretide Szeto-Schiller-31 (SS-31)) and inhibitors (apocynin, diphenylene iodonium (DPI), and 5-hydroxydecanoate (5-HD)) are shown in Table 1 . The following experimental groups were established: normoxia, hypoxia, hypoxia+NAC, hypoxia+DPI, hypoxia+apocynin, hypoxia+SS-31, hypoxia+5-HD, and hypoxia+apocynin+5-HD. All modulators were added to the medium 1 h prior to hypoxic stress.
9
Optimizing Hemocyte Culture Conditions
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To increase survival of the cultured C. farreri hemocytes, the medium was first optimized by supplementing fetal bovine serum (FBS, Hyclone) and CFS to the basic L15 medium (BM-L15, pH 7.2-7.4). BM-L15 medium consists of 13.7 g l -1 Leibovitz's 15 medium (Gibco), 20.2 g l -1 NaCl, 0.54 g l -1 KCl, 0.60 g l -1 CaCl 2 , 1 g l -1 MgSO 4 , 3.9 g l -1 MgCl 2 , 2 mmol l -1 glutamine (Sigma), 100 U ml -1 penicillin (Sigma), and 100 µg ml -1 streptomycin (Sigma). The different media used are summarized in Table 1. Hemolymph was seeded (0.2 ml; 5 × 10 4 cells) onto each of the 24well plates with 0.5 ml BM-L15 and supplements 1), and 3 wells were employed for each group. The primary culture was conducted in an SHP-080 biochemistry incubator at 23°C. The cultured cells were observed and photographed daily using a TS100 inverted microscope (Nikon). The cultured cells were digested with 0.25% trypsase at primary culture for 24 h and counted using a hemocytometer after staining with Trypan blue (Cao et al. 2003) (link) to determine the percentage of living cells (Table 1).
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