The in vitro experiments were conducted on macrophage cell line RAW 264.7 (American Type Culture Collection, Manassa, VA, USA). These cells were seeded onto the prepared specimens at a density of 105 cells/cm2 and maintained for 24 h in a humidified atmosphere of 5% CO2 at 37 °C in Dulbecco’s Minimal Essential Medium (DMEM, Sigma-Aldrich Co., St. Louis, MO, USA) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS, Life Technologies Corporation, Grand Island, NY, USA) and 1% (v/v) penicillin (10,000 units/mL)/streptomycin (10 mg/mL) (Sigma-Aldrich Co., St. Louis, MO, USA). The experiments were simultaneously performed in standard culture conditions and under pro-inflammatory stimulation (medium supplementation with 100 ng/mL Escherichia coli lipopolysaccharide (LPS, Sigma-Aldrich Co., St. Louis, MO, USA)).
Dulbecco s minimal essential medium dmem
Manufactured by Merck Group
Sourced in United States
Dulbecco's minimal essential medium (DMEM) is a cell culture medium formulated by Merck Group. It is a popular basal medium used to support the growth of a variety of cell types in vitro. The medium contains essential nutrients, amino acids, vitamins, and salts required for cell maintenance and proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Escherichia coli lipopolysaccharide lps, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Raw264, by American Type Culture Collection (1 mentions)
Lyovec, by InvivoGen (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Geneticin, by Thermo Fisher Scientific (1 mentions)
Penicillin g streptomycin, by Thermo Fisher Scientific (1 mentions)
Trypsin edta solution, by Merck Group (1 mentions)
2 7 dichlorofluorescein diacetate, by Merck Group (1 mentions)
Sterile filters 0.22 μm, by Merck Group (1 mentions)
Anti beclin 1, by Cell Signaling Technology (1 mentions)
Anti atg5, by Cell Signaling Technology (1 mentions)
Enhanced chemiluminescence ecl system kit, by Merck Group (1 mentions)
Dna fragmentation elisa kit, by Roche (1 mentions)
Cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Anti lc3b, by Cell Signaling Technology (1 mentions)
Polyvinylidene difluoride pvdf membrane, by Merck Group (1 mentions)
8 protocols using dulbecco s minimal essential medium dmem
1
In Vitro Macrophage Response to Inflammatory Stimuli
2
EBV-transformed AGS cell culture protocol
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HeLa cells (ATCC RR-B51S) were cultured in Dulbecco’s minimal essential medium (DMEM, Sigma-Aldrich), supplemented with 10% FCS (Gibco-Invitrogen), ciprofloxacin (10 μg/ml) and maintained in a 37°C incubator in 5% CO2. EBV converted AGS-Bx1 cell line (kindly provided by Alan Chiang, Hong Kong University, Hong Kong) [54 (link)] were cultured in Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12) (GIBCO-Invitrogen, Carlsbad, USA) supplemented with 10% Fetal Bovine Serum and 500 μg/ml Geneticin (GIBCO-Invitrogen). Plasmid transfection was performed using the JetPEI DNA transfection reagent (Polyplus transfection; Illkirch, France) as recommended by the manufacturer. Low molecular weight (LMW) Poly (I:C) was transfected using the transfection reagent LyoVec as Poly(I:C) (LMW) / LyoVec (InvivoGen; Toulouse, France) as per manufacturers guidelines. To induce the productive virus cycle AGS-Bx1 cells were cultured for 24 h in medium supplemented with 30 ng/ml TPA and 0.5 mM NaBu.
3
HeLa Cell Culture and Preparation
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HeLa cells were slowly defrosted, added to 5 mL of Dulbecco’s minimal essential medium (DMEM; Sigma–Aldrich, USA) in a falcon tube, and centrifuged for 4 min at 1200 rpm. The pellet was transferred into a mixture of DMEM solution supplemented with 10% of fetal bovine serum (GenClone, USA) and 1% of penicillin G/streptomycin (10,000 IU/mL to 10,000 μg/mL) (ThermoFisher Scientific, USA). About 5 mL of the mixture was seeded into a T-flask 25 cm2 for 24 h at 37 °C in a 5% CO2 incubator. The old medium was aspirated, and the cells were washed three times with 2 mL of PBS to remove debris [13 , 14 (link)]. About 1 mL of 0.25% trypsin–EDTA solution (Sigma–Aldrich, USA) was added to detach HeLa cells from the flask for cell calculation. About 1 × 105 HeLa cells/mL were seeded onto a 24-well tissue-culture plate in a 5% CO2 incubator for 24 h at 37 °C until a monolayer of cells reached (~ 90% confluence) a density of 2 × 105 cells/well [13 , 14 (link)]. The growth medium was diskharged, and the cells were washed twice with PBS to be used in Ld45E adhesion assays.
4
Oxidative Stress and Autophagy Modulation
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AgNO3 (CAS No. 7761-88-8), NaBH4 (CAS No.16940-66-2), N-acetyl-L-cysteine (NAC), 2, 7-dichlorofluorescein diacetate (CAS No. 4091-99-0), 5-bromo-2-deoxyuridine (BrdU; CAS No. 59-14-3) and Dulbecco’s Minimal Essential Medium (DMEM; CAS No. D5030-10L) were procured from Sigma Aldrich, USA. cDNA Synthesis kit was purchased from Fermentas, USA. Alexa 488-labelled antibodies were obtained from Molecular Probes. The Cell Titer-Glo Luminescent cell viability assay kit was procured from Promega Corp. (Madison, WI, USA). Anti-Atg5, Anti-Beclin1, anti-LC3B, Anti-pAMPK1α, Anti-GAPDH, Anti-β-actin, and rabbit anti-mouse horseradish peroxidase (HRP)-labeled secondary antibodies were procured from Cell Signaling Technology. Enhanced chemiluminescence (ECL) system kit, sterile filters (0.22 μm), and polyvinylidene difluoride (PVDF) membranes were procured from Millipore (MA, USA). DNA fragmentation ELISA kit was procured from Roche Diagnostics, Mannheim, Germany. All the chemicals used were of the highest purity grade. All the plastic wares used in the study for cell culture were procured from Thermo Fisher Scientific, USA.
5
Multifunctional Nanocarrier for Cancer Therapy
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Poly (ethyleneglycol) bis (3-aminopropyl ether) (PEG 1500); folic acid (FA); silver nitrate; poloxamer 407 (P-407); 1, 3-dicyclohexylcarbodiimide (DCC); 4-dimethylaminopyridine (DMAP); Dimethylsulfoxide (DMSO), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide (EDC/NHS), Dulbecco’s Minimal essential medium (DMEM), Fetal bovine serum (FBS), and MTT (3-(4, 5-Dimethylthiazol-2yl)-2, 5, diphenyltetrazoliumbromide) reagent were all purchased from Sigma-Aldrich, Bangalore, India. The National Centre for Cell Sciences in Pune, India, provided cancer cell lines such as HeLa (immortalized human cervical carcinoma) and Caco-2 (immortalized human epithelial colorectal adenocarcinoma) cells. Rhodamine B isothiocyanate, Toluidine Blue-O (TBO), Kaighn’s version of Ham’s F12 medium (F12K), Folate-free RPMI 1640 (FFRPMI), Fluorescence isothiocyanate (FITC), and EDTA/Trypsin were bought from BASF Corporation, Mumbai, India.
6
Propagation and Infection Kinetics of Nipah and Cedar Viruses
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The Bangladeshi strain of the Nipah virus (NIV) was obtained from the Viral Special Pathogens Branch of the CDC, was originally isolated from a fatal human infection in Bangladesh in 2004, and was passaged in Vero E6 cells a total of three times [16 (link)]. Cedar virus (CedPV) was obtained from CSIRO (Geelong, Australia) and was isolated from bat urine inoculated onto primary bat kidney cells, followed by passaging on Vero E6 cells (ATCC) [7 (link)]. Both viruses were propagated on Vero E6 cells at RML in Dulbecco’s minimal essential medium (DMEM) (Sigma) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 50 IU/mL penicillin, and 50 µg/mL streptomycin (Life Technologies, Carlsbad, CA, USA).
BHK-21 cells (CCL-10, ATCC, Manassas VA, USA) were propagated in 24-well tissue culture plates and inoculated with CedPV or NiV (or mock) with a MOI of 1.0 or 0.1 for 1 h, washed twice in DMEM, and incubated at 37 °C, 5% CO2 for the indicated amount of time.
Human lung blood microvascular endothelial cells (LBMVEC) (CC-2527, Lonza, Walkersville MD, USA) were maintained in an EGM-2 medium (Lonza). The cells were seeded in 24-well plates one day prior to inoculation with CedPV or NiV with a MOI of 0.1. At the indicated time points, supernatants were collected for virus quantitation and the cells were lysed in buffer RLT (Qiagen) to quantitate viral or cellular RNA.
BHK-21 cells (CCL-10, ATCC, Manassas VA, USA) were propagated in 24-well tissue culture plates and inoculated with CedPV or NiV (or mock) with a MOI of 1.0 or 0.1 for 1 h, washed twice in DMEM, and incubated at 37 °C, 5% CO2 for the indicated amount of time.
Human lung blood microvascular endothelial cells (LBMVEC) (CC-2527, Lonza, Walkersville MD, USA) were maintained in an EGM-2 medium (Lonza). The cells were seeded in 24-well plates one day prior to inoculation with CedPV or NiV with a MOI of 0.1. At the indicated time points, supernatants were collected for virus quantitation and the cells were lysed in buffer RLT (Qiagen) to quantitate viral or cellular RNA.
7
Colorectal Cancer Cell Line Treatment
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Experiments were performed on colorectal carcinoma cell lines DLD1 (CCL-221, ATCC, Sigma-Aldrich, St. Louis, MO, USA) and HCT116 (CCL-247, ATCC, Sigma-Aldrich, St. Louis, MO, USA) derived from clear cell renal cell carcinoma (ccRCC) (ECACC, 03112702, Sigma-Aldrich, St. Louis, MO, USA) and a non-cancerous cell line derived from EA.hy926 epithelial cells (ATCC, CRL-2922TM). The cell lines were cultured in an RPMI medium (Sigma-Aldrich, St. Louis, MO, USA) or Dulbecco’s minimal essential medium (DMEM, Sigma-Aldrich, St. Louis, MO, USA) with high glucose (4.5 g/L) and L-glutamine (300 μg/mL), supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA) and a penicillin/streptomycin mixture (Calbiochem, San Diego, CA, USA, penicillin 100U/mL, streptomycin 100 μg/mL). The cells were cultured in a water-saturated atmosphere at 37°C and 5% CO2. The experiments were performed with the cells in passage 5-15, and they were tested once a week for mycoplasma by double PCR. The cells were treated with paclitaxel (PTX, Selleckchem, Pittsburgh, PA, USA, 20 nmol/L), vincristine sulfate salt (vin, Sigma-Aldrich, St. Louis, MO, USA, 100 nmol/L), and a slow-releasing sulfide donor GYY4137 (GYY, Cayman Chemical, Ann Arbor, MI, USA, 10 µmol/L) (18 (link), 26 (link), 27 (link)) for 24 h.
8
Activation and Propagation of Rotavirus
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Rotavirus (strain SA-11) was activated by 10 µg/mL trypsin before propagated on MA-104 cells derived from kidney cells of African rhesus monkey. Cells were cultivated in tissue culture asks encompassing less than 5% CO 2 placed at 37°C in Dulbecco's Minimal Essential Medium (DMEM; Sigma-Aldrich Co., St.Louis, MO) along with additional 1% antibiotic antimycotic solution and 10% heat inactivated fetal bovine serum (A5955, Sigma-Aldrich Co., St. Louis, MO). Centrifugation of rotavirus was performed at 300 ×g for 5 mins in order to remove cell debris and make them pure. The supernatant was utilized as a stock suspension after ltration through 0.2 µm membrane. The stock virus contained 10 6 -10 7 TCID50 per mL and were kept at -80°C for future use.
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