The human retinal pigment epithelial cell line (ARPE‐19, CRL‐2302) was obtained from American Type Culture Collection (Manassas, VA, USA). ARPE‐19 cells were grown in DMEM/F‐12 (Lonza, Walkersville, MD, USA) supplemented with 10% foetal bovine serum (FBS; Atlanta Biologicals, Lawrenceville, GA, USA), 1% Glutamax (Lonza) and 1% penicillin–streptomycin (Lonza) at 37°C, 10% CO2. Primary human umbilical vein endothelial cells (HUVECs) were kindly provided by Ms. Case (Center for Excellence in Vascular Biology, Brigham and Women's Hospital, Boston, MA, USA). HUVECs from passages 3 through 6 were cultured in plastic flasks coated with 0.1% gelatin (Sigma‐Aldrich, St. Louis, MO, USA), in EGM‐2 (Lonza) with 20% FBS, SingleQuots supplements (Lonza), 1% Glutamax and 1% penicillin–streptomycin at 37°C, 10% CO2.
Glutamax
Manufactured by Lonza
Sourced in United States, Austria, Switzerland
GlutaMAX is a laboratory product manufactured by Lonza. It is a cell culture supplement that provides a stable source of L-glutamine, an essential amino acid required for cell growth and proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Glutamax, by Thermo Fisher Scientific (17 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (7 mentions)
Penicillin streptomycin, by Lonza (6 mentions)
Rpmi 1640, by Lonza (5 mentions)
B27 supplement minus vitamin a, by Thermo Fisher Scientific (3 mentions)
Glutamax, by R&D Systems (3 mentions)
B27 supplement minus vitamin a, by Lonza (3 mentions)
B27 supplement minus vitamin a, by R&D Systems (3 mentions)
Activin a, by R&D Systems (3 mentions)
Rpmi 1640 medium, by Merck Group (3 mentions)
Bone morphogenetic protein 4 (bmp4), by R&D Systems (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Pen strep, by Lonza (2 mentions)
Recombinant human egf, by Lonza (2 mentions)
Recombinant human fgf, by Lonza (2 mentions)
Nhdf neo, by Lonza (2 mentions)
Dulbecco modified eagle medium (dmem), by Lonza (2 mentions)
Docetaxel, by Merck Group (2 mentions)
Du145, by American Type Culture Collection (2 mentions)
Rpmi 1640, by PAN Biotech (2 mentions)
34 protocols using glutamax
1
Culture of Retinal Pigment Epithelial and Endothelial Cells
2
Expansion and Xenotransplantation of Human CD34+ Cells
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Human CB-CD34+ primary cells (Poetics, Lonza) were cultured in StemMACS HSC Expansion Medium (Miltenyi Biotech) supplemented with StemMACS HSC Expansion Cocktail (Miltenyi Biotech), containing stem cell factor (SCF), FLT3 ligand (FLT3L) and thrombopoietin (TPO). K562 cells were cultured in RPMI supplemented with 10% FBS, glutamax and pen/strep (all from Life Technologies). MS5 stromal cells were cultured in alpha MEM (Lonza) supplemented with 10% FBS, glutamax and pen/strep. All cells were maintained at 37°C in 5% CO2.
Female mice 6–8 weeks old, NOG strains were purchased from Harlan and maintained in specific pathogen-free (SPF) conditions prior to and during the in vivo experiments. The xenotransplant procedures in immunodeficient mice were approved by the Institutional Animal Welfare Committee of Biogem, and conformed in all their aspects to the institutional guidelines that comply with national and international laws and policies. The engraftment experiments were conducted according to the published guidelines for the welfare and use of animals [64 (link)]. All animals were monitored for a decrease in physical activity or other signs of disease or distress; severely ill animals were euthanized by CO2 asphyxiation.
Female mice 6–8 weeks old, NOG strains were purchased from Harlan and maintained in specific pathogen-free (SPF) conditions prior to and during the in vivo experiments. The xenotransplant procedures in immunodeficient mice were approved by the Institutional Animal Welfare Committee of Biogem, and conformed in all their aspects to the institutional guidelines that comply with national and international laws and policies. The engraftment experiments were conducted according to the published guidelines for the welfare and use of animals [64 (link)]. All animals were monitored for a decrease in physical activity or other signs of disease or distress; severely ill animals were euthanized by CO2 asphyxiation.
3
Colorectal Carcinoma Cell Viability
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Cell viability tests and study the cell cycle profiles were performed for observing a possible production of cell death in a tumor line of cells derived from colorectal carcinoma, called HCT116. This tumor line of HCT116 colorectal carcinoma cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco Invitrogen, Dublin, Ireland) supplemented 10% with decomplemented Heat-Inactivated Fetal Bovine Serum (FBS) (Gibco Invitrogen), with glutamax (BioWhittaker, Cologne, Germany) at 1% and with Penicillin/Streptomycin (BioWhittaker) at 1%. The cell culture was kept in an incubator at 37 °C and 5% of CO2.
4
Neonatal Dermal Fibroblast Expansion and Conditioned Medium
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Neonatal human dermal fibroblasts (NHDF-Neo; Lonza, Walkersville, MD, USA) were mixed with 40-mL fibroblast growth medium (FGMTM; Lonza) at a 1:1 ratio. Cells (4 × 105 cells) were seeded into a 75-cm2-T-flask and incubated overnight at 37°C under 5% CO2. The medium was replaced with Opti-modified Eagle’s medium (Opti-MEM; Gibco BRL, Rockville, MD, USA) to create low-serum culture conditions and the cells were cultured for another 24 h to reach approximately 90% confluence. Cells were then washed with Dulbecco’s phosphate buffered saline (PBS; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and treated with 0.15% trypsin (Gibco BRL) for 5 min at 37°C to detach cells from the surface of the culture flask. Medium was added to arrest trypsinization, and the cells were centrifuged for 5 min. The collected cells were resuspended in Opti-MEM and split into 75-cm2-T flasks. Cells were expanded until enough cells were prepared for experiments. To prepare DFCM, human dermal fibroblasts (passage 5) were cultured in 175-cm2-T flasks with Opti-MEM supplemented with 2-mM GlutaMAX, 2-ng/mL recombinant human EGF, and 2-ng/mL recombinant human FGF (all purchased from Lonza) without the addition of fetal bovine serum. The cells were incubated at 37°C in a 5% CO2 incubator for 24 h, and the medium was collected as DFCM.
5
Comparative Cell Line Maintenance
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Five different cell lines were used in this experiment. U373MG cells, currently reclassified as U251 (HTB-17, ATCC, Manassas, VA, USA) but here referred to with their original name for continuity with earlier publications from the group, were maintained in DMEM (Lonza, Basel, Switzerland) with 5% heat inactivated fetal bovine serum (FBS), 1% GlutaMAX (Gibco, Carlsbad, CA, USA), and gentamycin. HCE cells (kindly provided by Arto Urtti from the University of Helsinki and the University of Eastern Finland, Kuopio, Finland) and SW480 cells (CCL-228, ATCC, Manassas, VA, USA) were maintained in the same medium as the U373MG cell line. Vero cells (CCL-81, ATCC, Manassas, VA, USA) were maintained in M199 medium (Gibco), 5% heat inactivated FBS, and gentamycin. Raji cells (CCL-86, ATCC, Manassas, VA, USA) were maintained in RPMI 1640 (Lonza) with 10% heat inactivated FBS, 1% GlutaMAX, and gentamycin.
6
T-cell Activation and Cytokine Production
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PBMCs were either not or stimulated with anti-CD3/anti-CD28 T-cell expander beads (Invitrogen Dynal, Oslo, Norway) at different ratios, i.e. 1 cell / 0.1 bead, 1 cell / 0.5 bead, 1 cell / 1 bead for 6 h in human culture medium (HCM; RPMI-1640 with GlutaMAX, 10% heat-inactivated pooled human serum and 1% penicillin and streptomycin) (Lonza, Breda, Netherlands) with Golgistop (BD). Then cells were stained with AmCyan-labeled anti-CD3 (BD), Pacific Blue-labeled anti-CD4 (BD), APC-Cyanin 7 (APC-Cy7)-labeled anti-CD8 (BD), APC-labeled anti-CD45RO (BD) and PE-Cy7-labeled anti-CCR7 (R&D Systems) antibodies and a live–dead marker ViaProbe (7-aminoactinomycin D; 7AAD; BD). Upon fixation with FACS lysing solution (BD) and permeabilization using FACS permeabilizing solution 2 (BD), cells were stained intracellular using PE-labeled anti-CD69 (BD) and FITC-labeled anti-IL-2 (BD). Percentages CD69-expressing and IL-2 producing CD4+ T cell subsets were evaluated upon measuring the samples on a BD FACSCanto II flow cytometer (BD). Data were analyzed by Kaluza™ software (Beckman Coulter).
7
Prostate Cancer Cell Line Cultivation
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Human cell lines PC3, DU145, and LNCaP were obtained from American Type Culture Collection (ATCC, Rockville, MD). LAPC4 were a kind gift from Prof. A. Cato (Institute of Toxicology and Genetics, Karlsruher Institut für Technologie, Germany). Docetaxel‐resistant PC3‐DR and DU145‐DR were previously established by Puhr et al.14 All cells were cultured in Roswell Park Memorial Institute 1640 (RPMI‐1640) (PAN Biotech, Aidenbach, Germany) containing 10% (v/v) fetal calf serum (PAN Biotech), 1% (v/v) penicillin/streptomycin and 1% (v/v) GlutaMAX (both from Lonza, Vienna, Austria). LNCaP were supplemented with 1% (v/v) 4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid (HEPES) (Sigma, Vienna), 1% (v/v) d ‐glucose (Sigma), 1% (v/v) Na‐pyruvate (Lonza) and LAPC4 with 100 nmol/L dihydrotestosterone (Sigma). PC3‐DR and DU145‐DR were cultured in the presence of 12.5 nmol/L Docetaxel (Sigma). The authenticity of all cell lines was validated via short tandem repeat profiling.
8
Colony Formation Assay for Stem and Progenitor Cells
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Cells sorted by flow cytometry or bulk population single cells were plated in methylcellulose (Methocult from Stem Cell Technology, France) mixed with one of the following media: Stem cell medium I: 20 ng/ml EGF (PeproTech, USA), 10 ng/ml bFGF (PeproTech, USA), 1× B27 (Gibco) and 1× PS (Lonza) in 1× Defined Keratinocyte-SFM (Gibco); Stem Cell medium II: 20 ng/ml EGF, 20 ng/ml bFGF, 1× B27, 1× PS and 1× Glutamax (Gibco) in DMEM-F12 (Gibco); Pancreatic medium: Pancreatic culture media with pancreatic cell culture supplement and PS and RPMI medium: 10% FCS, 1× PS and 1× Glutamax in RPMI 1640 (Lonza) according to manufacturers' protocol. In brief, single cell suspensions were added to media with Methocult, mixed and seeded at a density of 1000 cells/well in a non-adhesive 24 well plate (Grenier Bio-One, Germany). Two weeks post plating, the colonies were stained with 150 µl 0.4 mg/ml Thiazolyl Blue Tetrazolium Bromide (MTT) (Sigma-Aldrich) at 37°C for 4 hours or overnight. Uniform colonies of minimum 50 µm were scanned using the GelCount machine and the number and sizes were quantified using GelCount software (both from Oxford Optronix, UK).
9
Isolation of Bone Marrow-Derived Macrophages
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For the scratch test, bone marrow–derived macrophages (BMDM) were isolated from femurs and tibias of 8 week-old wild-type C57BL/6J mice as described by Suen et al. (1999 ) [24 (link)] and Baruzzi et al. [25 (link)]. Briefly, cells were cultured in DMEM with Glutamax (Lonza) supplemented with 15% FBS, 10% L929-cell conditioned medium (LCM) as a source of colony-stimulating factor-1, 100 U/ml penicillin, and 100 μg/ml streptomycin (BMDM complete medium), and cultured at 37°C/5% CO2 in 75 cm2 flasks. After 24 h, the non-adherent cells were removed, counted, plated on bacteriological (non tissue-culture-treated) plastic dishes at a concentration of 1×105/ml, and cultured in BMDM complete medium.
10
Detailed Protocol for Cell Proliferation Assay
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RPMI-1640, PBS, Glutamax, and Hepes were obtained from Lonza (Austria). Fetal calf serum was purchased from PAA (GE Healthcare, UK), MEM w/o leucine, 0.25% Trypsin/EDTA from Gibco, and YoYo-1 fluorescent dsDNA staining from Molecular Probes (Life Technologies, UK), and tritiated Leucine from Perkin Elmer (Waltham, MA). Cyclosporine A was purchased from Calbiochem (San Diego, CA) and dissolved in ethanol to 8.3 mM stock solution. The GenElute Mammalian total RNA kit and general laboratory chemicals were from Sigma Aldrich (St. Louis, MO), the Cell Titer 96 AqueousOne solution (MTS) cell proliferation assay was from Promega (Madison, WI). RT2 Profiler PCR Array System, including the cDNA synthesis kit, and SYBR green were from SABiosciences (Qiagen Nordic). Chemicals for validation of gene expression were from Applied (Life Technologies, UK). Plastic ware for cell culture was from Nunc (Thermo Scientific), gels and buffers for protein electrophoresis from Life Technologies, HRP-conjugated antibodies from Dako (DK), and chemiluminescent super-signal substrate from Thermo Scientific.
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