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7 protocols using mem non essential amino acids

1

Cytokine and Cytolytic Granule Production Assay

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To measure cytokine and cytolytic granule production, cells were isolated by draining the auricular, axillary, brachial, cervical, and inguinal LNs (pooled LNs) of mice fed either a normal diet or rodent chow containing 1% (w/w) ABC for 1 week. The cells were seeded in 96-well plates at a density of 5.0 × 105 cells/well in 100 μL RPMI-1640 medium containing 10% FBS, 2 mM l-glutamine, 1 mM sodium pyruvate, 1× MEM non-essential amino acids (Nacalai Tesque), 10 mM HEPES, 0.05 mM 2-mercaptoethanol, penicillin (100 units/mL), and streptomycin (100 μg/mL). The plates were then incubated at 37 °C in a humidified atmosphere of 95% air/5% CO2 for 4 h by stimulating with Cell Activation Cocktail (phorbol 12-myristate-13-acetate and ionomycin) (BioLegend) and monensin (BioLegend).
For intracellular staining, the cultured cells were subjected to the following treatment. Briefly, the cell surfaces were initially stained with anti-mCD8a Ab and anti-mouse/humanCD44 Ab for 30 min at 4 °C. The cells were then fixed with 4% paraformaldehyde for 10 min at room temperature and permeabilized with 0.5% (v/v) Triton X-100 for 10 min at 4 °C. Finally, all samples were incubated with anti-cytokine Ab or cytolytic granule Ab for 30 min at 4 °C.
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2

Cell Culture Protocols for Various Cell Lines

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A172, CHO, SH-SY5Y, H4, and U87 were purchased from the American Type Culture Collection (Rockville, MD, USA). A172 and H4 cells were cultured in DMEM (Nacalai Tesque, Inc.; Kyoto, Japan) supplemented with 10% fetal bovine serum (FBS; Life Technologies Corp.; Grand Island, NY, USA) and a mixture of penicillin, streptomycin, and amphotericin B (PSA; Nacalai Tesque, Inc.; Kyoto, Japan). SH-SY5Y and CHO were cultivated in DMEM/Ham’s F-12 (Nacalai Tesque, Inc.; Kyoto, Japan) supplemented with 10% FBS and PSA. U87 was cultivated in EMEM (FUJIFILM Wako Pure Chemical Corp.; Osaka, Japan) supplemented with 10% FBS, PSA, sodium pyruvate (Nacalai Tesque, Inc.; Kyoto, Japan), and MEM non-essential amino acids (Nacalai Tesque, Inc.; Kyoto, Japan). All cell lines were maintained at 37 °C in 5% CO2 incubator (MCO-175; Sanyo Electric Co., Ltd.; Osaka, Japan).
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3

Culturing Ovarian and Breast Cancer Cell Lines

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Mouse ovarian cancer cell line OV2944-HM-1 (HM-1) and breast cancer cell line 4T1 were purchased from the Japanese Collection of Research Bioresources and the American Type Culture Collection, respectively. The HM-1 and 4T1 cell lines were maintained in MEMa and RPMI-1640, respectively (Fujifilm Wako Pure Chemical Industries, Osaka, Japan). All culture media were supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA), 100 U/mL penicillin (Nacalai Tesque Inc., Kyoto, Japan), 100 µg/ml streptomycin (Nacalai Tesque Inc.), 0.1 mM MEM nonessential amino acids (Nacalai Tesque Inc.), and maintained at 37°C with 5% CO2.
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4

Naive CD8 T Cell Activation and Glutamine Deprivation

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Naive CD8 T (CD44lowCD62Lhigh) cells were prepared using a Naive CD8+ T-cell Isolation kit (cat#130-096-543; Miltenyi Biotec, San Diego, CA, USA). Naive CD8 T cells (1.5 × 106) were stimulated with immobilized anti-TCR-β mAb (3 μg/ml, H57-597; BioLegend) and anti-CD28 mAb (1 μg/ml, 37.5; BioLegend) for 2 days in the presence of IL-2 (10 ng/ml, Pepro Tech). The cells were then transferred to a new plate and further cultured in the presence of IL-2 (10 ng/ml). The cells were cultured in RPMI 1640 with l-glutamine (cat#189-02025; Wako Chemicals) supplemented with 10% fetal bovine serum, 2 mM l-glutamine (16948-04; Nacalai Tesque, Kyoto, Japan), 1 mM sodium pyruvate (cat#06977-34; Nacalai Tesque), 1% MEM nonessential amino acids (cat#06344-56; Nacalai Tesque), 10 mM HEPES (cat#15630-080; Thermo Fisher Scientific, Waktham, MA, USA), 55 μM 2-Mercaptoethanol (cat#21985-023; Thermo Fisher Scientific), and 1% penicillin-streptomycin (cat#26253-84; Nacalai Tesque). For glutamine-deprived conditions, the cells were cultured in RPMI 1640 without l-glutamine (cat#183-02165; Wako Chemicals) supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, 1% MEM nonessential amino acids, 10 mM HEPES, 55 μM 2-Mercaptoethanol, and 1% penicillin-streptomycin.
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5

Cell Apoptosis Evaluation Protocol

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RPMI 1640 medium was purchased from Nissui Pharmaceutical Co., Ltd. (Tokyo, Japan). Opti-modified Eagle’s medium (Opti-MEM) and fetal bovine serum (FBS) were obtained from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Sodium chloride, sodium bicarbonate, potassium chloride, Tris-HCl, ethylenediaminetetraacetic acid (EDTA), ammonium chloride, and glucose were purchased from Wako Pure Chemicals Industries, Ltd. (Osaka, Japan). Monothioglycerol, MEM non-essential amino acids, and penicillin-streptomycin-glutamine mixed solution were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). The 20 bp ladder was purchased from Takara Bio (Otsu, Japan). Propidium iodide, R848, and ovalbumin were obtained from Merck KGaA (Darmstadt, Germany). Alexa Fluor 488 annexin V/ Dead Cell Apoptosis kit with Alexa Fluor 488 annexin V and PI for Flow Cytometry was purchased from Invitrogen (San Diego, CA, USA). All other chemicals were of the highest grade available and were used without further purification.
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6

Cell Culture Protocols for Diverse Cell Lines

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C2C12, HepG2, 293T, COS7, and LLC cells were cultured in DMEM (Nacalai Tesque) containing 15% (for C2C12 cells) or 10% FCS (Invitrogen), 1× MEM non‐essential amino acids (Nacalai Tesque), and 100 U/mL penicillin/streptomycin (PS; Wako). To induce myoblast differentiation, C2C12 cells were cultured in DMEM containing 2% horse serum (Cosmo Bio), 1× MEM non‐essential amino acids, and PS.
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7

Mouse Cancer Cell Line Maintenance

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Mouse breast cancer cell line 4T1, mouse colon cancer cell line CT26, mouse lung cancer cell line 3LL, mouse melanoma cell line B16, and mouse fibrosarcoma cell line MCA205 were purchased from the American Type Culture Collection. 4T1 was designated as 4T1-ATCC (4T1A) and the highly immunogenic sub-clone of 4T1A, established in 2015, was named 4T1-Sapporo (4T1S)11 (link). All cell lines were maintained in RPMI-1640 (Fujifilm Wako Pure Chemical Corporation). Culture medium was supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich), 100 U/ml penicillin (Nacalai Tesque), 100 µg/ml streptomycin (Nacalai Tesque), 0.1 mM MEM nonessential amino acids (Nacalai Tesque), and maintained at 37°C with 5% CO2. Mycoplasma contamination of cell lines was checked using the MycoAlert® Mycoplasma Detection Kit (Lonza Group), according to the manufacturer’s instructions; no contamination was found.
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