The gastric cancer cell line GCIY was used to investigate microbe–host interactions. The adhesion assay was done as described previously (Shibahara-Sone et al., 2016 (link)) with minor modifications. Briefly, cells of WT or mutant #1476 were collected by centrifugation (3,000 g, 10 min, 4°C), washed once with Eagle's minimal essential medium (Nissui, Osaka, Japan) without antibiotics and containing 10% fetal bovine serum (Thermo Fisher Scientific), and resuspended in the same medium. GCIY cells were seeded into 96-well culture plates and grown to reach 2×103 or 1×104 cells/well. A sheet of GCIY cells (2×103 or 1×104 cells/well) was rinsed with fresh medium and incubated with the WT or #1476 suspension (1×105, 1×106, or 1×107 CFU/well) or medium (negative control) at 37°C for 30 min in humidified air containing 5% CO2. The cell sheet was then rinsed in fresh medium three times, acidified fresh medium (pH 4.5) was added, and the cell sheet was incubated for 4.5 h. The acid-treated cell sheet was then rinsed in fresh medium three times, and cell morphology was observed by microscopy. The acid-treated cell sheet was then incubated in fresh (non-acidified) medium overnight, and viable cell counts and morphological changes were investigated.
Eagle s minimal essential medium
Manufactured by Nissui Pharmaceutical
Sourced in Japan
Eagle's minimal essential medium is a cell culture medium designed to support the growth and maintenance of various cell types. It provides the necessary nutrients and components required for cellular proliferation and survival. The medium is formulated to maintain a stable and balanced environment for cell cultures.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Bovine calf serum (bcs), by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Fujifilm (1 mentions)
Dulbecco s modified eagle s medium, by Fujifilm (1 mentions)
Tryptose phosphate broth, by BD (1 mentions)
Cell banker 1, by Nippon Zenyaku Kogyo (1 mentions)
Dnase 1, by Takara Bio (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Nacalai Tesque (1 mentions)
Dnase 1, by Nacalai Tesque (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Nissui Pharmaceutical (1 mentions)
Percoll solution, by Cytiva (1 mentions)
Ribavirin, by Merck Group (1 mentions)
Ma104, by American Type Culture Collection (1 mentions)
96 well plate, by Corning (1 mentions)
8 protocols using eagle s minimal essential medium
1
Gastric Cancer Cell Line Adhesion Assay
2
Immune Stimulants Preparation Protocol
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LPS, NiCl2, CoCl2, ZnCl2, PdCl2, NiSO4, poly(I:C), and zymosan A were dissolved in water and AcD was dissolved in ethanol and diluted with Eagle’s minimal essential medium (Nissui, Tokyo, Japan). The final concentration of ethanol was adjusted to 0.1% (v/v). All stimulants are soluble at the concentrations used in this study.
3
Preparation and Propagation of Chicken Embryo Fibroblasts
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Chicken embryo fibroblasts (CEFs) were prepared from 10-day-old fertilized eggs (Iwamura Hatchery Co., Ltd., Shibata, Japan) as described previously [32 (link)]. CEFs were cultured in Eagle’s minimal essential medium (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) supplemented with 10% bovine calf serum (Sigma-Aldrich, St. Louis, MO, USA), 10% tryptose phosphate broth (Difco Laboratories, Detroit, MI, USA), 0.03% L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.1% NaHCO3. DF-1 cells, a chicken fibroblast cell line, were cultured with 0.5 mL of Dulbecco’s modified Eagle’s medium (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), containing 10% fetal bovine serum, and incubated at 39 °C under 5% CO2. Viruses were reconstituted by transfecting BAC DNA into CEFs as described previously [33 (link)]. Viruses were propagated on CEFs for seven passages, and virus stocks were frozen in Cell Banker 1 (Nippon Zenyaku Kogyo Co., Ltd., Fukushima, Japan) and titrated on CEFs using plaque assays as described previously [34 (link),35 (link)].
4
Isolation of Bacterial Genomic DNA from L929 Cells
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The bacterial cells were inoculated to L929 cells and incubated in Eagle’s minimal essential medium (Nissui, Japan) containing 1% fetal bovine serum and 2 mM L-glutamine at 34°C for 7–10 days as previously described (Fujita, 2008 ). After centrifugation at 800 rpm for 5 min to remove L929 cell debris, the supernatants were centrifuged at 14,000 rpm for 10 min to recover bacterial cells. The obtained pellets were suspended in phosphate-buffered saline (PBS, pH 7.2). The cell suspensions were treated with DNase I (Takara Bio, Japan; 2,000 U/ml at a final concentration) for 1 h at 37°C to digest cell-free DNA. DNase I was inactivated by incubation at 65°C for 5 min, and EDTA (Nacalai Tesque, Japan) was added to the solutions at a final concentration of 2 mM. Bacterial genomic DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Japan) according to the manufacturer’s instructions.
5
Cell Culture Protocols for BLV, BIV, and BFV
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FLK-BLV cells (which are persistently infected with BLV), CC81 (a feline cell line transformed by mouse sarcoma virus), and CC81-BLU3G and CC81-GREMG cells (derivatives of CC81) [5 (link)] were cultured at 37 °C in 5% CO2 in Dulbecco’s modified Eagle’s Medium (DMEM) (Thermo Fisher, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA). BIV-infected bovine embryonic spleen cells and BFV-infected fetal bovine muscle cells were cultured at 37 °C/5% CO2 in Eagle’s minimal essential medium (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) supplemented with 10% FBS, as described previously [17 (link), 18 (link)].
6
Isolation of Murine Immune Cells
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Mice thymocytes, splenocytes, and liver mononuclear cells (MNCs) were prepared using the previously described methods [30] . Briefly, cells were obtained from each organ by using repetitive pipetting and a 200-gauge stainless steel mesh, and the cells were suspended in Eagle's minimal essential medium (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 5 mM HEPES and 2% heat-inactivated newborn calf serum. After being washed once with the medium, the cells from the thymus were used as thymocytes. After washing them with the medium, the cells from spleens were further resuspended in erythrocyte lysis solution (155 mM NH 4 Cl, 10 mM KHCO 3 , 1 mM Na-EDTA, and 17 mM Tris-HCl (pH 7.3)). After being washed once with the medium, the cells were used as splenocytes. Liver cells were further resuspended in 15 mL of 35% percoll solution (Amersham Pharmacia Biotech) containing 100 U/mL heparin, and were centrifuged at 300 × g for 15 min to remove hepatocytes. The pellet was resuspended in erythrocyte lysis solution (the same as the splenocyte preparation). After being washed once with the medium, the cells were used as liver MNCs.
7
Isolation of Hepatic Mononuclear Cells
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Mice were killed on the days indicated in the figures after infection. Hepatic mononuclear cells were isolated using a previously described method (16 (link)). Briefly, the liver was removed, pressed through 200-gauge stainless steel mesh, and suspended in Eagle’s minimal essential medium (Nissui Pharmaceutical Co., Tokyo, Japan) supplemented with 5 mM HEPES and 2% heat-inactivated newborn calf serum. After washing once with medium, the cells were fractionated by centrifugation in 15 ml of 35% Percoll solution (Pharmacia Fine Chemicals, Piscataway, NJ) for 15 min at 424.8 g. The pellet was resuspended in erythrocyte lysing solution (155 mM NH4Cl, 10 mM KHCO3, 1 mM EDTA-Na and 170 mM Tris, pH 7.3). The splenocytes were obtained by forcing the spleen through 200-gauge stainless steel mesh. Splenocytes were used after erythrocyte lysing.
8
Antiviral Potential of Herbal Extracts
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MA104 (Rhesus monkey kidney cell line) was purchased from ATCC (USA) and cultured in monolayer cultures using Eagle’s minimal essential medium (Nissui Pharmaceutical, Japan) containing 10% heat-inactivated fetal bovine serum (FBS; Equitech-Bio, USA) and 2 M glutamine in 5% CO2 at 37°C. Cells were exposed to RV strain SA11 and harvested after 2 freeze-thaw cycles and stored in aliquots at -80°C. Virus titer was determined by a fluorescent-focus assay with 96-well plates (Corning Incorporated, USA).
V. jatamansi was powdered and extracted with 95% ethanol and refluxed. The combined extract was filtered and evaporated (yield 20.7% (w/w)). Albiziae Cortex and Ziziphi Spinosae Semen were powdered, diluted in distilled water, and evaporated with 15.2% (w/w) and 25.8% (w/w) respectively. Junci Medulla was extracted with aqueous ethanol (95%, 1.5 L, v/v) and concentrated (4.5% (w/w)). The extracts were mixed at a ratio of 5:3:5:1 with prepared V. jatamansi Jones.
Ribavirin was purchased from Sigma-Aldrich Inc. (USA). Cells were divided into five groups: normal group (without treatment), low group (10 μM V. jatamansi Jones), medium group (20 μM V. jatamansi Jones), high group (30 μM V. jatamansi Jones). The morphological change was observed using a microscope.
V. jatamansi was powdered and extracted with 95% ethanol and refluxed. The combined extract was filtered and evaporated (yield 20.7% (w/w)). Albiziae Cortex and Ziziphi Spinosae Semen were powdered, diluted in distilled water, and evaporated with 15.2% (w/w) and 25.8% (w/w) respectively. Junci Medulla was extracted with aqueous ethanol (95%, 1.5 L, v/v) and concentrated (4.5% (w/w)). The extracts were mixed at a ratio of 5:3:5:1 with prepared V. jatamansi Jones.
Ribavirin was purchased from Sigma-Aldrich Inc. (USA). Cells were divided into five groups: normal group (without treatment), low group (10 μM V. jatamansi Jones), medium group (20 μM V. jatamansi Jones), high group (30 μM V. jatamansi Jones). The morphological change was observed using a microscope.
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