Seven-week-old male BALB/c mice (n = 10) were killed by cervical dislocation, and spleens were removed and crushed to obtain splenocytes. NK cells were isolated from splenocytes by negative selection using an NK cell isolation kit (110-115-818, Miltenyi Biotec, Bergisch Gladbach, North Phine-Westphalia, Germany) according to the manufacturer’s instructions [31 (link)]. Isolated NK cells were cultured at a density of 5 × 106 cells/mL in T-25 flasks in Roswell Park Memorial Institute-1640 medium (Cytiva, Marlborough, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Waltham, MA, USA), 1% L-glutamine (Gibco), 1% Antibiotic-Antimycotic (Gibco), 50 µM β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA) and 50 ng/mL recombinant human IL-15 protein (R&D systems, Minneapolis, MN, USA). Fresh medium was added to the cells on days 4, 5, and 6. Cells were maintained in a humidified atmosphere containing 5% CO2 and 95% air at 37 °C.
1640 medium
Manufactured by Cytiva
Sourced in United States
1640 medium is a cell culture medium designed for the cultivation of a variety of cell types. It provides essential nutrients and growth factors to support cell growth and proliferation. The medium is formulated to maintain the optimal pH and osmolarity for cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin p s, by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Cytiva (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Recombinant human il 15 protein, by R&D Systems (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by GE Healthcare (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Hepes, by Merck Group (1 mentions)
Sodium bicarbonate, by Merck Group (1 mentions)
Su1498, by Thermo Fisher Scientific (1 mentions)
Vascular endothelial growth factor (vegf), by Thermo Fisher Scientific (1 mentions)
Matrigel, by BD (1 mentions)
Hec 1b, by Thermo Fisher Scientific (1 mentions)
9 protocols using 1640 medium
1
Isolation and Culture of Mouse NK Cells
2
Cell Line Validation and Culture
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Two cell lines (T24 and J82) were used in the present study, which were validated by performing DNA fingerprinting analysis (Korean Cell Line Bank; Korean Cell Line Research Foundation; Fig S1 ). The cells were cultured in Roswell Park Memorial Institute-1640 medium (Cytiva) at 37°C with 5% CO2. The culture medium weas supplemented with 10% fetal bovine serum (GE Healthcare), 10 mM HEPES (Sigma-Aldrich; Merck KGaA), 2.0 g/l sodium bicarbonate (Sigma-Aldrich; Merck KGaA), 1 mM sodium pyruvate (Thermo Fisher Scientific, Inc.) and 100 U/100 µg/ml penicillin-streptomycin (Thermo Fisher Scientific, Inc.).
3
Bacterial Adhesion Assay in Intestinal Cells
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The adhesion ability of the bacteria was measured in HT-29 cells and hPSC-derived hIECs. The cell lines were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Cytiva, USA) with 10% fetal bovine serum (FBS; HyClone, USA) and 1% penicillin/streptomycin (PS; Gibco, USA) at 37°C in a humidified incubator with 5% CO2.
The hPSC line (H9) was purchased from the WiCell Research Institute (Madison, USA). The hPSCs were cultured on Mitomycin C-treated mouse embryonic fibroblasts (MMC-MEF) using hPSC medium [Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Gibco), 10% serum replacement (SR, Gibco), 1% PS (Gibco), 1%GlutaMAX (Gibco), 0.1% β-mercaptoethanol (Gibco), and 8 ng/ml basic fibroblast growth factor (bFGF; R&D Systems, USA)]. The hPSCs were passaged every week to newly prepared MMC-MEF [19 (link)]. All experiments were approved by the Public Institutional Review Board designated by the Ministry of Health and Welfare (P01-201409-ES-01).
The hPSC line (H9) was purchased from the WiCell Research Institute (Madison, USA). The hPSCs were cultured on Mitomycin C-treated mouse embryonic fibroblasts (MMC-MEF) using hPSC medium [Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Gibco), 10% serum replacement (SR, Gibco), 1% PS (Gibco), 1%GlutaMAX (Gibco), 0.1% β-mercaptoethanol (Gibco), and 8 ng/ml basic fibroblast growth factor (bFGF; R&D Systems, USA)]. The hPSCs were passaged every week to newly prepared MMC-MEF [19 (link)]. All experiments were approved by the Public Institutional Review Board designated by the Ministry of Health and Welfare (P01-201409-ES-01).
4
Bacterial Adhesion Assay in Intestinal Cells
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The adhesion ability of the bacteria was measured in HT-29 cells and hPSC-derived hIECs. The cell lines were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Cytiva, USA) with 10% fetal bovine serum (FBS; HyClone, USA) and 1% penicillin/streptomycin (PS; Gibco, USA) at 37°C in a humidified incubator with 5% CO2.
The hPSC line (H9) was purchased from the WiCell Research Institute (Madison, USA). The hPSCs were cultured on Mitomycin C-treated mouse embryonic fibroblasts (MMC-MEF) using hPSC medium [Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Gibco), 10% serum replacement (SR, Gibco), 1% PS (Gibco), 1%GlutaMAX (Gibco), 0.1% β-mercaptoethanol (Gibco), and 8 ng/ml basic fibroblast growth factor (bFGF; R&D Systems, USA)]. The hPSCs were passaged every week to newly prepared MMC-MEF [19 (link)]. All experiments were approved by the Public Institutional Review Board designated by the Ministry of Health and Welfare (P01-201409-ES-01).
The hPSC line (H9) was purchased from the WiCell Research Institute (Madison, USA). The hPSCs were cultured on Mitomycin C-treated mouse embryonic fibroblasts (MMC-MEF) using hPSC medium [Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Gibco), 10% serum replacement (SR, Gibco), 1% PS (Gibco), 1%GlutaMAX (Gibco), 0.1% β-mercaptoethanol (Gibco), and 8 ng/ml basic fibroblast growth factor (bFGF; R&D Systems, USA)]. The hPSCs were passaged every week to newly prepared MMC-MEF [19 (link)]. All experiments were approved by the Public Institutional Review Board designated by the Ministry of Health and Welfare (P01-201409-ES-01).
5
Angiogenesis Inhibition Protocol
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Medium M199, Dulbecco’s modified Eagle’s medium (DMEM), RPMI (Roswell Park Memorial Institute)-1640 medium, fetal bovine serum (FBS) were obtained from Hyclone Laboratories (South Logan, utah, USA). VEGF and SU1498 were ordered from PeproTech (Rocky Hill, NJ, USA) and Calbiochem (Merck KGaA, Darmstadt, Germany). Matrigel were obtained from BD Bioscience (Bedford, MA, USA). All antibodies were ordered from Cell Signaling Technology (Danvers, MA, USA) or Abcam (Louis Park, MN, USA). CA4 was synthesized as described14 (link)29 (link). For the in vitro studies, CA4 was dissolved in dimethyl sulfoxide (DMSO as vehicle) at a concentration of 10 mM and then subsequently diluted in culture medium.
6
Linc01194 Expression in Human Endometrial Carcinoma
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The expression of linc01194 in 4 human EC cells lines (Ishikawa, HEC-1B, HEC-1A, and KLE), purchased from Jennio Biotech, was detected. Roswell Park Memorial Institute (RPMI) 1640 medium (HyClone Laboratories Inc., Logan, UT, USA) was utilized to culture Ishikawa cells, while Dulbecco’s Modified Eagle Medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA) was utilized to culture HEC-1B cells, McCoy’s 5A medium was utilized to culture HEC-1A cells and Ham’s F-12 treatment medium was utilized to culture KLE cells. All culture media contained 10% fetal bovine serum (FBS) and penicillin/streptomycin. Cells were cultured in an incubator containing 5% CO2 at 37°C. All cells were identified by short tandem repeat (STR) analysis and subjected to mycoplasma contamination tests.
7
Reagents and Antibodies for Cell Signaling Analysis
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Dimethyl sulfoxide (DMSO), bicinchoninic acid (BCA), trichloroacetic acid (TCA), sulforhodamine B (SRB), and catalase were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA), Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), trypsin-EDTA solution (1X), antibiotic-antimycotic solution (100X) and phosphate-buffered saline (PBS) (1X) were purchased from HyClone Laboratories, Inc. (South Logan, UT, USA). Anti-p-AMPKα, anti-AMPKα, anti-p-ACC, anti-ACC, anti-p-mTOR, anti-mTOR, anti-p-4EBP1, anti-4EBP1, anti-p-eIF4E, anti-eIF4E, anti-p-P70S6K1, anti-P70S6K1, anti-p-RPS6, anti-RPS6, anti-rictor, anti-p-Akt, anti-Akt, and anti-E-cadherin were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-PKC-α, anti-p-Rac1, anti-Rac1, anti-PCNA, anti-β-actin, and all secondary antibodies were purchased from Santa Cruz Biotechnology. (Santa Cruz, CA, USA). Anti-p-rictor was purchased from Millipore (Temecula, CA, USA). Anti-p-PKC-α, anti-F-actin, and anti-Ki-67 were purchased from Abcam (Cambridge, MA, USA). Gefitinib was purchased from Selleckchem (Houston, TX, USA) and rapamycin was purchased from Tocris Bioscience (Bristol, UK).
8
Cell Culture Protocols for Cancer and Endothelial Cells
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Human breast cancer cell line MD-MBA-231 cells were cultured in Leibovitz’s L-15 medium (Gibco®; Thermo Fisher Scientific), murine fibroblast cell line L929 cells were cultured in Roswell Park Memorial Institute 1640 medium (HyClone Laboratories, Inc., Logan, UT, USA), and human umbilical vein endothelial cells (HUVECs) were cultured in endothelial cell medium (ScienCell Research Laboratories, Carlsbad, CA, USA), with all the medium being supplemented with 10% FBS, penicillin (100 units/mL), and streptomycin (100 mg/mL).
9
Human NSCLC Cell Maintenance Protocol
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Human NSCLC cell lines (A549 and H1975) were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were maintained in Roswell Park Memorial Institute-1640 medium (Hyclone Laboratories, Logan, UT, USA) supplemented with 10% fetal bovine serum (Hyclone Laboratories), 100 IU/mL penicillin, and 100 µg/ mL streptomycin (Welgene, Daegu, Korea) in a humidified atmosphere of 5% CO2 at 37 °C. All in vitro experiments were performed under two conditions: one was a normal condition as described above and the other was a hypoxic condition that was maintained at 1% O2, 5% CO2 and 94% nitrogen.
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