Ovarian cancer cells from one patient with ovarian cancer (54.2 years old) were collected with written informed consent on May 2015 in Weifang People's Hospital (Weifang, China). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Biowhittaker; Lonza Group, Ltd., Basel, Switzerland) supplemented with 10% fetal bovine serum (FBS; Biowhittaker; Lonza Group, Ltd.) at 37°C and 5% CO2 for 24 h. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy mice and cultured as described in a previous study (24 (link)). SKOV3, OVCAR3, CAOV-3 and A2780 cells were purchased from the Cell Bank of the Chinese Academy of Science (Shanghai, China). All tumor cells were cultured in DMEM supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin under standard culture conditions (5% CO2, at 37°C) for 12 h.
Biowhittaker
Manufactured by Lonza
Sourced in Switzerland, United States, Belgium, Italy, Germany, United Kingdom
The BioWhittaker is a laboratory equipment product manufactured by Lonza. It is designed for cell culture and biopharmaceutical applications. The core function of the BioWhittaker is to provide a controlled environment for the cultivation and growth of cells.
Automatically generated - may contain errors
Lab products found in correlation
L glutamine, by Thermo Fisher Scientific (5 mentions)
Penicillin, by Thermo Fisher Scientific (5 mentions)
Streptomycin, by Thermo Fisher Scientific (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Penicillin streptomycin, by Lonza (4 mentions)
Amphotericin b, by Thermo Fisher Scientific (4 mentions)
Streptomycin, by Lonza (3 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (3 mentions)
40 m cell strainer, by BD (2 mentions)
Countess automated cell counter, by Thermo Fisher Scientific (2 mentions)
Mcf 7, by American Type Culture Collection (2 mentions)
Hyclone, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Merck Group (2 mentions)
Puromycin, by Merck Group (2 mentions)
Penicillin, by Lonza (2 mentions)
L glutamine, by Lonza (2 mentions)
Penicillin g, by Merck Group (2 mentions)
Penicillin streptomycin, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Hemocytometer, by Lonza (2 mentions)
80 protocols using biowhittaker
1
Culturing Ovarian Cancer and PBMC Cells
2
MERS-CoV Infectious Virus Assay
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Baby hamster kidney cells (BHK-21) were obtained from American Type Culture Collection (ATCC CCL-10). Human liver-derived Huh-7 cells were kindly provided by R. Bartenschlager (University of Heidelberg, Germany). Cells were grown in BioWhittaker (Lonza) or Dulbecco’s modified Eagle medium (DMEM) with 2% glutamine, 1% non-essential amino acids (Sigma) and 5% (BHK-21) or 8% (Huh-7) fetal bovine serum (FBS) (HyClone, ThermoFisher). The bronchial epithelial cell line Calu-3 2B4 [54 (link)] was kindly provided by CT Tseng (University of Texas Medical Branch, USA). Calu-3 cells were grown in BioWhittaker (Lonza) 1 g/L glucose medium with 2% glutamine, 1% non-essential amino acids (Sigma) and 15% fetal bovine serum (FBS) (HyClone, ThermoFisher).
Virus titers were determined on Huh-7 cells following standard procedures and using closed flasks or plates sealed in plastic bags. For plaque assays, infected cells were overlaid with DMEM containing 0.6% low-melting agarose and 2% FBS, and at 72 hpi, cells were fixed with 10% formaldehyde and stained with 0.1% crystal violet. All work with MERS-CoV infectious viruses was performed in biosafety level 3 facilities at CNB-CSIC or the University of Iowa according to the guidelines set forth by each institution.
Virus titers were determined on Huh-7 cells following standard procedures and using closed flasks or plates sealed in plastic bags. For plaque assays, infected cells were overlaid with DMEM containing 0.6% low-melting agarose and 2% FBS, and at 72 hpi, cells were fixed with 10% formaldehyde and stained with 0.1% crystal violet. All work with MERS-CoV infectious viruses was performed in biosafety level 3 facilities at CNB-CSIC or the University of Iowa according to the guidelines set forth by each institution.
3
Primary Hippocampal Neuron Culture and Transfection
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Primary hippocampal neuronal cultures were obtained from newborn (P1‐P3) rats as previously described (Banker & Cowan, 1977 ; Kaech & Banker, 2006 ). Cells were grown in 12‐well plates on glass coverslips in Neurobasal‐A medium (Gibco, Paisley, Scotland), pH 7.5, at 37°C in 5% CO2.
Cells were transfected using either calcium phosphate or lipofectamine transfection. Transfections with calcium phosphate were performed after 7–8 days in vitro, using the ProFection® Mammalian Transfection System Calcium Phosphate Kit following a protocol according to the manufacturer's recommendations with slight modifications described previously (Truckenbrodt et al,2018 ).
Lipofection was performed after 2–6 days in vitro. Coverslips with cells were placed into 400 μl of DMEM (BioWhittaker, Lonza, Verviers, Belgium) complemented with 10 mM MgCl2 and incubated for 30 min at 37°C in 5% CO2. 50 μl of a transfection mix containing 2 μl of Lipofectamine® 2000 reagent (Invitrogen, Carlsbad, CA, USA) and 1 μg of plasmid DNA in Opti‐MEM® (Gibco, Paisley, Scotland) medium were added per coverslip. After 20‐min incubation at 37°C in 5% CO2, cells were washed three times with DMEM (BioWhittaker, Lonza, Verviers, Belgium) complemented with 10 mM MgCl2 and placed into original wells with Neurobasal‐A medium (Gibco, Paisley, Scotland).
Cells were transfected using either calcium phosphate or lipofectamine transfection. Transfections with calcium phosphate were performed after 7–8 days in vitro, using the ProFection® Mammalian Transfection System Calcium Phosphate Kit following a protocol according to the manufacturer's recommendations with slight modifications described previously (Truckenbrodt et al,
Lipofection was performed after 2–6 days in vitro. Coverslips with cells were placed into 400 μl of DMEM (BioWhittaker, Lonza, Verviers, Belgium) complemented with 10 mM MgCl2 and incubated for 30 min at 37°C in 5% CO2. 50 μl of a transfection mix containing 2 μl of Lipofectamine® 2000 reagent (Invitrogen, Carlsbad, CA, USA) and 1 μg of plasmid DNA in Opti‐MEM® (Gibco, Paisley, Scotland) medium were added per coverslip. After 20‐min incubation at 37°C in 5% CO2, cells were washed three times with DMEM (BioWhittaker, Lonza, Verviers, Belgium) complemented with 10 mM MgCl2 and placed into original wells with Neurobasal‐A medium (Gibco, Paisley, Scotland).
4
Establishment of EGFR-mutant NSCLC Cell Lines
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The human NSCLC cell lines HCC827 and HCC4006, which harbor EGFR activating mutations (16 (link),17 (link)), were purchased from the Chinese Academy of Sciences (Shanghai, China). Both cell lines were cultured in RPMI-1640 medium (BioWhittaker®; Lonza Group, Ltd., Basel, Switzerland) supplemented with 10 mM HEPES, 1 mM L-glutamine, 100 U/ml penicillin/streptomycin (BioWhittaker®; Lonza Group) and heat inactivated 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and grown at 37°C in a 5% CO2 atmosphere. Gefitinib (Iressa; AstraZeneca, Macclesfield, UK) was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at a concentration of 10 mM and stored at −20°C. Gefitinib-resistant HCC827R and HCC4006R cells were established by initially culturing with 1 µM Gefitinib in DMEM plus 10% FBS for 6 weeks. Subsequently, a 2-µM concentration of Gefitinib was used to treat the surviving cells for 8 weeks and 5 µM for another 8 weeks. Eventually, the Gefitinib-resistant NSCLC cell lines were successfully established by culturing the cells in 10 µM Gefitinib.
5
MDCK Cell Culture Protocol
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Madin Darby canine kidney (MDCK) cells and Madin Darby canine kidney (MDCK-SIAT) cells were obtained from St. Jude Children’s Research Hospital, USA and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (BioWhittaker, Lonza, Cologne, Germany) supplemented with 5% inactivated fetal bovine serum (FBS; Gibco BRL Life Technologies, Grand Island, NY, USA) and 1% antibiotic-antimycotic mixture (BioWhittaker, Lonza, Cologne, Germany) and grown at 37 °C and 5% CO2.
6
Cell Culture Conditions for Human Cell Lines
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HCT-116, A549, 22Rv1, C4-2B, LNCap, R1-AD1, R1-D567, PC3 and PNT1A were cultured with RPMI 1640 media (Sigma Life Science, Gillingham, UK). HepG2 and Caco-2 were cultured with DMEM media (BioWhittaker®, Lonza, Basel, Switzerland). All the media were supplemented with 10% FCS (Fetal Calf Serum, First Link UK Ltd., Wolverhampton, UK) and 10% L-glutamine (gibco®, Life Technologies, Paisley, UK; 200 mmol, 100 mL). Caco-2 cell line was maintained in DMEM media (BioWhittaker®, Lonza, Basel, Switzerland) containing 20% FCS (Fetal Calf Serum, First Link UK Ltd., Wolverhampton, UK) and 10% L-glutamine (gibco®, Life Technologies, Paisley, UK; 200 mmol, 100 mL). Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2. Cell lines were authenticated by provider (Human colorectal adenocarcinoma cell line, ATCC). No additional authentication of cells was performed.
7
Isolation of human CD14+ monocytes
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Human CD14+ cells were isolated from healthy volunteers. PBMCs were separated by density gradient centrifugation on Lymphoprep (Axis-Shield, Oslo, Norway), and the CD14+ cells were isolated by magnetic sorting using CD14 MACS microbeads (Miltenyi Biotech, Bergisch Gladbach, Germany) according to the manufacturer's protocol. The cells were cultured in DMEM medium (Lonza BioWhittaker, Thermo Fisher Scientific cat. nr: 11635220) supplemented with 10% human AB serum (pooled from five healthy volunteers), L-glutamine (5 mM), sodium pyruvate (5 mM), and glucose (2.5 g/L) (Gibco, Thermo Fisher Scientific).
8
Cell Line Characterization and Maintenance
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Jurkat, Human embryonic kidney cells 293 (HEK293T) and THP-1 were purchased from ATCC. Epstein Barr Virus (EBV)-transformed B cell lines were obtained from the Lupus Family Registry and Repository (OMRF) with IRB approval. EBV cell lines were selected using genotype data corresponding to the rs10499197 variant. Genotypes were verified by Sanger sequencing. Cell lines were maintained in RPMI 1640 medium supplemented with 10% FBS, 1X penicillin-streptomycin antibiotic mixture (Atlanta Biologicals Inc., GA), 2mM Lglutamine (Lonza™ BioWhittaker™, Walkersville, MD), and 55μM ß-mercaptoethanol. The following antibodies were used in the study: anti-GAPDH (Cell Signaling Inc., Danvers, MA); anti-A20 (GeneTex Inc., Atlanta, GA); anti-Histone H3 (acetyl K27) (Abcam, Cambridge, MA); normal Rabbit IgG (EMD Millipore, Burlington, MA). All stock laboratory chemicals were from Sigma Aldrich(St.Louis,MO).
9
Metal-Induced PBMC Proliferation Assay
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Samples were collected after informed consent and before PT, to prevent sensitisation. Peripheral Blood Mononuclear Cells (PBMC) were isolated with Lymphocyte Separation Medium and resuspended in RPMI-1640 containing 10% Fetal Bovine Serum. Non-toxic concentrations of challenge metals (0,1 mM; 0,01 mM) were selected by a dose response curve. 2 × 105 cells/well were seeded in triplicate in 96-well plates with or without Chromium (III) chloride (CrCl3), Chromium powder (Cr), Nickel (II) chloride (NiCl2), Nickel nanopowder (Ni), Cobalt powder (Co), Titanium powder (Ti), and Molybdenum nanopowder (Mo). Ni and Cr, the main sensitiser metals, were analyzed both in soluble and particulate form. Phytohemagglutinin (PHA 0,01 mg/ml) was used as control. After 5 days, cells were pulsed overnight with 3H-thymidine (1 μCi/well) and proliferation was assessed by scintillation counting. Results were expressed as Stimulation Index (SI = mean cpm-treated /mean cpm-untreated cultures). Culture media and supplements were purchased from Biowhittaker, (Lonza, Treviglio, Italy), chemicals from Sigma Aldrich (Milan, Italy).
10
Protein Extraction from Glioblastoma Cells
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Glioblastoma cells were washed with 1x Phosphate buffered saline (PBS) (BioWhittaker®), trypsinised (Trypsin:EDTA, Sigma-Aldrich®) and centrifuged at 1,200 rpm for 4 minutes. The supernatant was removed and protein was extracted from the resulting pellet in Protease inhibitor (1:100, Sigma-Aldrich®) and RIPA buffer (1M Tis pH 7.4, 5M NaCl, 1% NP-40, 50 mM NaF, 0.5M EDTA, 0.1% SDS, 0.5% Na-deoxycholate). Resuspended cells in RIPA were left on ice for 20 minutes before centrifugation at 4°C at 12,000 rpm for 10 minutes. Supernatant was isolated, quantified using a BCA™ Protein Assay kit (Thermo Fisher Scientific) and stored at –20°C.
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