HEK293 and HepG2 cells were cultured with MEM medium (Corning) supplemented with 10% fetal calf serum. Twenty-four hours after seeding, transfection was conducted using Lipofectamine 3000 (Invitrogen), following the manufacturer’s protocol. In the 96-well-plate, each well were transfected with firefly luciferase vectors, the control renilla luciferase vector pRL-CMV, and/or the HNF4A1 expression vector. Twenty-four hours after transfection, cells were harvested for dual-luciferase assay using Dual-Glo™ luciferase assay system (Promega) and GloMax Luminometer (Promega), following the manufacturer’s protocol. The ratios of firefly/renilla luciferase activities were calculated as the normalized reporter activity, with the control values set at 1.0. For treatment of the G4-specific ligand, pyridostatin (PDS, Sigma) in aqueous solution was added 18 h after transfection of HEK293 cells63 (link), and HEK293 cells were harvested for dual-luciferase assay 6 h after PDS treatment.
Mem medium
Manufactured by Corning
Sourced in United States, China
MEM medium is a cell culture medium designed for the maintenance and growth of various cell types. It provides the necessary nutrients, salts, and other components to support cell survival and proliferation in vitro. The core function of MEM medium is to create an optimal environment for cell cultivation and experimentation.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640 medium, by Corning (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Coronavirus hcov nl63 nr 470, by BEI Resources (2 mentions)
Sars cov 2 strain wa1, by BEI Resources (2 mentions)
Dmem medium, by Corning (2 mentions)
Dmem f12 medium, by Corning (2 mentions)
L glutamine, by Corning (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Pyridostatin pds, by Merck Group (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Dual glo luciferase assay system, by Promega (1 mentions)
Glomax luminometer, by Promega (1 mentions)
Mcl 1, by Santa Cruz Biotechnology (1 mentions)
Gefitinib, by Cayman Chemical (1 mentions)
Basal medium eagle, by Merck Group (1 mentions)
P akt ser473, by Cell Signaling Technology (1 mentions)
Cyto c, by Santa Cruz Biotechnology (1 mentions)
Grp78, by Santa Cruz Biotechnology (1 mentions)
C parp, by Santa Cruz Biotechnology (1 mentions)
Cyclin b1, by Santa Cruz Biotechnology (1 mentions)
19 protocols using mem medium
1
Dual-Luciferase Assay for HNF4A1 Activity
2
Gefitinib-Induced Apoptosis Pathway
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DPT was prepared by Professor Goo Yoon according to previous reports [42 ]. RPMI1640 medium, phosphate-buffered saline, fetal bovine serum, penicillin/streptomycin, and trypsin were purchased from Hyclone (Logan, UT, USA). MEM medium was purchased from Corning (Corning, NY, USA). DMSO, MTT, and Basal Medium Eagle were purchased from Sigma Chemical Company (St. Louis, MO, USA). Gefitinib was purchased from Cayman Chemical (Ann Abor, MI, USA). Primary antibodies against cyclin B1, cdc2, p21, p27, β-actin, GRP78, CHOP, DR4, DR5, Bcl-XL, Mcl-1, Bid, Bad, cyto c, α-tubulin, COX4, apoptotic protease activating factor-1 (Apaf-1), and c-PARP were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against p-EGFR (Tyr1068), EGFR, p-GSK-3β (Ser9), GSK-3β, p-ERK (Thr202/Tyr204), ERK, p-AKT (Ser473), and AKT were purchased from Cell Signaling Biotechnology (Beverly, MA, USA).
3
Cultivation and Characterization of SARS-CoV-2 and HCoV-NL63
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As established viral target cells infected by HCoV-NL63 and SARS-CoV-2, Calu-3 human metastatic lung epithelial adenocarcinoma (HTB-55), Caco-2 human colorectal epithelial adenocarcinoma (HTB-37) and Vero normal epithelial monkey kidney (CCL-81) cells (all from ATCC, Manassas, VA, USA) were maintained according to published standard procedures [15 (link)–18 (link)]. In brief, all cells (Calu-3, Caco-2 and Vero) were cultured in Eagle’s Minimum Essential Medium (MEM) medium (Corning, Manassas, VA) supplemented with 10% bovine calf serum (BCS, HyClo ne™ Laboratories, Logan, UT). Coronavirus HCoV-NL63 (NR-470) and its genomic RNA (NR-44105) were obtained from BEI Resources (NIAID, NIH). SARS-CoV-2 strain WA1 (NR-52281; BEI Resources) was propagated in Vero cells unless specified otherwise [6 (link)]. For viral stocks, cells were infected at a multiplicity of infection (MOI) of 0.01 and cultured for 48 h. At that point, cells were harvested, homogenized, subjected to a single freeze-thaw cycle, and then combined with the culture supernatant followed by centrifugation (3000 rpm, 10 min). The viral titers of the final supernatant (after serial dilution) was determined by plaque forming assay. All work with SARS-CoV-2 was performed under BSL3 conditions in a facility with negative pressure and PPE that included Tyvek suits and N95 masks for respiratory protection.
4
PFOS Cytotoxicity Evaluation in Cell Lines
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PFOS (potassium salt; >98% pure) was purchased from MAYA-R (Jiaxing, China). Dimethyl sulfoxide (DMSO) and the cell counting kit-8 (CCK-8) were purchased from Vicmed (Busan, Korea). MEM medium was purchased from Corning (Shanghai, China). DMEM/F12 medium and RPMI 1640 medium were purchased from KeyGEN BioTECH (Nanjing, China). Fetal bovine serum was purchased from ExCell Bio. HiScript® II Q RT SuperMix for RT-PCR and AceQ® RT-PCR were obtained from Vazyme (Nanjing, China). Rosiglitazone and GW9662 were purchased from MedChemExpress (Shanghai, China). pcDNA-PPARγ and siRNAs were obtained or synthesized by GenePharma (Shanghai, China). Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) was purchased from Invitrogen and used for transient transfection.
5
Virus Propagation in Cell Lines
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The SwPV (NVSL catalogue number 002‐PDV) was amplified in PK‐15 cells (porcine kidney), whereas the SVA strain HI/2012‐NADC40 (kindly provided by Drs. Lager and Buckley – USDA ARS) was propagated in swine testis (ST) cells. The BVDV strain Singer (NVSL catalogue number 140‐BDV) was amplified in Madin–Darby bovine kidney (MDBK) cells. Cell lines were cultured at 37°C with 5% CO2 in MEM medium (Corning) supplemented with 10% fetal bovine serum (Seradigm), 2 mM l‐glutamine (Corning), 1% Antibiotic‐Antimycotic 100X (Gibco), and gentamicin (50 μg/ml; Corning).
When the cytopathic effect was observed in more than 90% of the monolayers, the flasks were submitted to a freeze‐thaw cycle, followed by centrifugation at 1200× g for 5 min. The clarified viral stock was titrated using limiting dilution assay, and the titre was calculated using the method of Reed and Muench (1938 (link)). Viral stocks were titrated in triplicates and stored at −80°C until use. The viral stocks’ titers were 106.8, 109.5, and 107.5 tissue culture infectious dose (TCID50/ml) for SwPV, SVA, and BVDV.
When the cytopathic effect was observed in more than 90% of the monolayers, the flasks were submitted to a freeze‐thaw cycle, followed by centrifugation at 1200× g for 5 min. The clarified viral stock was titrated using limiting dilution assay, and the titre was calculated using the method of Reed and Muench (1938 (link)). Viral stocks were titrated in triplicates and stored at −80°C until use. The viral stocks’ titers were 106.8, 109.5, and 107.5 tissue culture infectious dose (TCID50/ml) for SwPV, SVA, and BVDV.
6
Cell Culture Protocols for MDCK and A549 Cells
7
Cell Culture Protocols for HT-29 and HEK 293 Cells
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HT-29 was kindly provided by Dr. Julio Tapia (Cell Transformation Laboratory, Faculty of Medicine, University of Chile). HT-29 and HEK 293 cells were cultured in high glucose DMEM (Corning Inc., Tewksbury, MA) with 10% Fetal Bovine Serum (FBS, Hyclone Pittsburgh, PA), Penicillin/Streptomycin (1%). HEK 293T cells were cultured in MEM medium (Corning Inc., Tewksbury, MA) 10% SFB and Penicillin/Streptomycin (1%).
8
Breast Cancer Cell Lines Characterization
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Human mammary epithelial (HMEC) primary cells were purchased from Lonza and UACC3199 cells were obtained from University of Arizona Cancer Center. Other breast cancer cells were obtained from the American Type Culture Collection (Manassas, VA). Cells were tested negative for mycoplasma contamination and validated for species and unique DNA profile using short tandem repeat analysis by the provider or us. Twenty two breast cancer cell lines were cultured and maintained in the specified media. Breast epithelium cell lines 184A1 and 184B5 as well as primary cell line HMEC were cultured in MEGM medium (Lonza CC-3150). Breast cancer cell lines CAMA1 and MCF7 (with additional 0.01mg/ml bovine insulin) were cultured in MEM medium (Corning 10–010) with 10% FBS. HS578T was cultured in DMEM medium (Gibco 11995–065) with 10% FBS and 0.01mg/ml bovine insulin. All other cell lines were cultured in RPMI 1640 medium (Gibco 11875–093) with 10% FBS and 1% HEPES. Total RNA and DNA were isolated from cell lines using the RNeasy Mini Kit (Qiagen, Montgomery, MD, USA) and GentraPuregene Cell Kit (Qiagen, Montgomery, MD, USA), respectively. The integrity of RNAs was validated by bio-analyzer at the University of Chicago Genomics Core Facility. RNAs with minimum RNA integrity number of 8 were applied to cDNA synthesis.
9
Cultivation of Human Cancer Cell Lines
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Human breast cancer cell lines (MDA-MB-231, MDA-MB-468, and Hs578T), DLBCL cell line (Pfeiffer, Karpas 422, WSU-DLCL2 and SU-DHL6), and HEK293T were purchased from Cobioer Biosciences Co., Ltd (Nanjing, China). Pfeiffer, Karpas 422, WSU-DLCL2 and SU-DHL6 cell lines were cultured in RPMI-1640 medium (Corning, Midland, NY, USA) with 10% FBS (VivaCell, Shanghai, China), and MDA-MB-231, MDA-MB-468 and HEK293T were cultured in DMEM medium (Corning) with 10% FBS (VivaCell, Shanghai, China), Hs578T were cultured in MEM medium (Corning, Midland, NY, USA) with 10% FBS(VivaCell, Shanghai, China) and supplemented with 1% penicillin/streptomycin (v/v).
10
Cultivation and Propagation of Zebrafish and Carp Cells
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Zebrafish embryonic fibroblast (ZF4) cells were purchased from American Type Culture Collection (ATCC number CRL-2050). Epithelioma papulosum cyprini (EPC) cells and SVCV (ATCC: VR-1390) were gifts by professor Jun-Fa Yuan (Huazhong agricultural university, Wuhan, Hubei, China). The SVCV was propagated in EPC cells at 28°C. The virus production was conducted according to previously described protocols (30 (link)). ZF4 and EPC cells were cultured at 28 °C in a 5% CO2 incubator in DMEM/F-12 medium (Corning, USA) and MEM medium (Corning, USA) with 10% fetal bovine serum (Gibco, Australia), respectively.
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