The following cell lines were purchased from the American Type Culture Collection (ATCC Rockville, MD, USA): CaSki, (cervical cancer), MDA-MB-231 (breast cancer), and SK-Lu-1 (lung cancer). They were cultured in RPMI-1640 medium (GIBCO, Invitrogen Corp., Grand Island, NY, USA) containing 5% Newborn Calf Serum (NCS, GIBCO, Invitrogen Corp., Grand Island, NY, USA) with red phenol supplemented by benzylpenicillin. All cultures were stored in a humidified atmosphere with 5% CO2 at 37 °C. All cell-based assays were performed using cells in the exponential growth phase.
Newborn calf serum ncs
Manufactured by Thermo Fisher Scientific
Sourced in United States
Newborn Calf Serum (NCS) is a cell culture supplement derived from the blood of newborn calves. It provides a rich source of proteins, growth factors, and other essential nutrients to support the growth and maintenance of a variety of cell types in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Pharm lyse, by BD (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Mda mb 231, by American Type Culture Collection (1 mentions)
Caski, by American Type Culture Collection (1 mentions)
Sk lu 1, by American Type Culture Collection (1 mentions)
Lornoxicam, by Merck Group (1 mentions)
Tenoxicam, by Merck Group (1 mentions)
Meloxicam, by Merck Group (1 mentions)
Piroxicam, by Merck Group (1 mentions)
Probenecid, by Merck Group (1 mentions)
Heparin, by MP Biomedicals (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Amphotericin b, by Merck Group (1 mentions)
Ibuprofen, by Merck Group (1 mentions)
Transferrin, by Merck Group (1 mentions)
Fibronectin, by BD (1 mentions)
Plus reagent, by Thermo Fisher Scientific (1 mentions)
Lipofectamine, by Thermo Fisher Scientific (1 mentions)
Jetprime, by Polyplus Transfection (1 mentions)
12 protocols using newborn calf serum ncs
1
Cell Culture Protocols for Cancer Research
2
In vitro blood-brain barrier assay
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Celecoxib, diclofenac, lornoxicam and diazepam were a kind gift of Dr. Maierhofer (AGES, PharmMed, Austria), whereas ibuprofen (I1892, SigmaAldrich, Austria), meloxicam (M3935, SigmaAldrich, Austria), piroxicam (P5654, SigmaAldrich, Austria), tenoxicam (T0909, SigmaAldrich, Austria), carboxyfluorescein (21877, Fluka, Switzerland), probenecid (P8761, SigmaAldrich, Austria) and verapamil (94837, Fluka, Switzerland) were purchased from commercial sources. Iscove’s modified Dulbecco’s medium (IMDM), Ham reg. nutrient mixture F12 (Ham’sF-12), newborn calf serum (NCS), l-glutamine and penicillin/streptomycin were obtained from Invitrogen Life technologies (GibcoTM, Carlsbad, CA). Heparin and collagen solution (predominantly collagen I, 150703) were purchased from MP Biomedicals (Irvine, CA). Amphotericin B, transferrin, 8-aminopyrene-1,3,6-trisulfonate (APTS) and gelatine were from SigmaAldrich (Austria), whereas dextran (av. MW 6000) was from Fluka (Switzerland). Fibronectin was obtained from BD Biosciences (Bedford, MA) as well as Transwell inserts (FalconTM) and six-well plates (FalconTM). Basal endothelial and astrocyte media and components for primary rat endothelial cells and astrocytes were provided from Biopredic Int. (France). Inorganic salts and all other reagents were of analytical grade.
3
Vav Protein Expression and Focus Formation
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NIH3T3 cells were cultured in DMEM supplemented with 10% Newborn Calf Serum (NCS; Invitrogen and PAA). For the focus formation assays, cells were cultured for 12-14 days after transfection with Lipofectamine and Reagent Plus (Invitrogen); medium was replaced every 2 or 3 days and was supplemented or not with JNK (SP600125) or P38 (SB203580) kinases inhibitors (Calbiochem). Foci were scored after Giemsa staining. Stable NIH3T3 cell lines expressing the various Vav proteins and vector control were selected in the presence of 1 mg/ml neomycin after transfection. Neomycin-resistant colonies were scored after 10-12 days of selection.
H358, H441 and A549 cell lines were maintained in RPMI and DMEM respectively, supplemented with 10% Foetal Bovine Serum (FBS; PAA). H358 and A549 cell lines were transfected with pcDNA constructs by using the Amaxa or JetPrime (Polyplus) following recommendation.
SiRNA control or targeting Vav1 were purchased from Dharmacon and transfected using JetPrime reagent (Polyplus).
H358, H441 and A549 cell lines were maintained in RPMI and DMEM respectively, supplemented with 10% Foetal Bovine Serum (FBS; PAA). H358 and A549 cell lines were transfected with pcDNA constructs by using the Amaxa or JetPrime (Polyplus) following recommendation.
SiRNA control or targeting Vav1 were purchased from Dharmacon and transfected using JetPrime reagent (Polyplus).
4
Immunoblot Analysis of JNK Signaling
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Anti-phospho JNK (Cat. No. 9251S), anti-JNK (Cat. No. 9252S) and phospho-c-Jun (Cat. No. 9146) were purchased from Cell Signalling Technologies (Beverley, MA, USA). Anti-a-tubulin (Cat. No. T5168) and anti-Flag M2 (Cat. No. F-3165) were purchased from Sigma (Poole, U.K.). Anti-HA (Cat. No. Sc805) was from Santa Cruz Biotechnology (Santa Cruz, California, USA). A rabbit polyclonal antiserum against full-length rat MS1 (1–375) was produced at Cambridge Research Bioscience (Cambridge, UK). Anti-rabbit and anti-mouse horseradish peroxidase (HRP) conjugates were purchased from GE Healthcare Life Sciences (Amersham, UK). Dulbecco’s Modified Eagle Medium (DMEM), penicillin, streptomycin, collagenase, heat-inactivated fetal calf serum (FCS), new-born calf serum (NCS) and optiMEM were purchased from Invitrogen (Paisley, UK). Pancreatin (Cat. No. P3292) and gelatin (Cat. No. G2500) were purchased from Sigma (Poole, UK). The inhibitors SP600125 and actinomycin D were from CN Biosciences Inc (San Diego, CA, USA).
5
Single-cell Isolation from Murine Tissues
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Single‐cell suspensions of BM cells were prepared by flushing the leg bones (one tibia and one femur per mouse) with RPMI‐1640 supplemented with 2% Newborn Calf Serum (NCS; Thermo Fisher Scientific) plus antibiotic‐antimycotic. Single‐cell suspensions of spleens were prepared by dissociating the tissues and filtered through a 40‐μm cell strainer (SPL Life Sciences Co., Ltd. Pocheon‐si, Korea). Peripheral blood was harvested, and complete blood count analysis was performed using VetScan® HM2 analyzer (Abaxis, Inc. Union City, CA, USA). Red blood cells (RBCs) were removed by using RBC lysing buffer (Sigma‐Aldrich, Saint Louis, MO, USA). Peripheral blood mononuclear cells were collected using Histopaque®‐1083 (Sigma‐Aldrich). Tumors were weighed and dissected mechanically and then digested with 400 units mL−1 collagenase D (Sigma‐Aldrich) and 200 μg mL−1 DNase I (Sigma‐Aldrich).
6
Detailed Characterization of Murine Mesothelioma Cell Lines
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Murine mesothelioma cell lines AB1 (CBA, Cat# CBA-0144, RRID: CVCL_4403), AB1-HA (CBA, Cat# CBA-1374, RRID: CVCL_G361) and AE17 (CBA, Cat#CBA-0156, RRID: CVCL_4408) were derived as previously described (14 (link), 15 (link)). Cell lines were maintained in RPMI 1640 (ThermoFisher Scientific, Scoresby VIC, Australia) supplemented with 20 mM HEPES, 0.05 mM 2-mercaptoethanol, 100 units/mL penicillin (CSL, Melbourne VIC, Australia), 50 μg/mL gentamicin (David Bull Labs, Kewdale VIC, Australia), 10% Newborn Calf Serum (NCS; ThermoFisher Scientific, Scoresby VIC, Australia) and 50 mg/mL of geneticin for AB1-HA only (G418; Life Technologies). Cells were cultured for a minimum of 4 passages after thawing before inoculation into mice. Cell lines were validated yearly by flow cytometry for MHC-I molecules H2‐Kb (consistent with C57BL/6) and H2‐Kd (consistent with BALB/c), and for fibroblast markers E-cadherin, epithelial cell adhesion molecule, and platelet-derived growth factor receptor α (negative). All cell lines were tested for Mycoplasma spp., every 3 months by PCR and found to be negative.
7
Culturing Endothelial and HEK293 Cells
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HRMECs were obtained from Angio-Proteomie (Peabody, MA, United States). HRMECs were cultured in endothelial cell medium (ECM) supplemented with 5% Newborn Calf Serum (NCS; Life Technologies), 20 μg/ml of ECGS, 100 μg/ml streptomycin, and 100 U/ml penicillin in a T-25 flask coated with 8.5 μg/ml of BPF at 37°C with 5% CO2. When the cells became 90% confluent, they were subcultured at a 1:3 ratio. HEK293 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, United States) and cultured in DMEM supplemented with 10% NCS and incubated at 37°C with 5% CO2.
8
Isolation of Lymphocyte Subsets
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For single-cell suspensions, thymus and spleen were crushed and filtered through 80μm stainless steel mesh (Sefar Ltd., UK) in fluorescence-activated cell sorting (FACS) buffer (phosphate-buffered saline (PBS) containing; 2% heat-inactivated fetal calf serum (FCS) (Life technologies); and 5 mM ethylenediaminetetraacetic acid (EDTA) (Life technologies)). For spleen, red blood cells were removed by gradient centrifiguation at 1600rpm for 25 min using 4 mL of Lymphocyte Separation Medium (Fischer). For IEL preparations, faecal material was flushed from lumen of small intestine with ice-cold PBS using a gavage needle. Fatty tissues, vasculature and Peyer’s patches were removed, followed by longitudinal opening and 60 min agitation in RPMI-1640 10% Newborn calf serum (NCS) (Life technologies) with 5 mM EDTA (Life technologies) at 37 °C. Cells were subsequently passed through an autoclaved column containing 0.7 g of nylon wool (Polysciences, USA), equilibrated with RPMI-1640 with 38 mM HEPES (Life technologies). IELs were enriched on a discontinuous Percoll gradient (40%/80% isotonic Percoll), before use and stained as described below.
9
Antioxidant and Anti-Inflammatory Assays
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Aluminum chloride (AlCl3) and Folin–Ciocalteu phenol reagent were purchased from Fisher Scientific, India. Ascorbic acid was purchased from Qualigens Fine Chemicals, India. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) was from Wako Pure Chemicals, Japan. Ferric chloride (FeCl3) was from Rasayan Laboratories, India. 3T3-L1 preadipocyte and RAW 264.7 cells were obtained from the American Type Culture Collection (ATCC). Bacteria were isolated from clinical samples, identified and supplied by Manipal Teaching Hospital, Phulbari Pokhara. Growth medium (Dulbecco’s modified Eagle’s medium [DMEM]), penicillin and streptomycin antibiotic solution, phenol red-free DMEM, fetal bovine serum (FBS), and newborn calf serum (NCS) were from Life Technologies Corporation, Waltham, MA, USA. Components of the differentiation media (3-isobutyl-1methylxanthine [IBMX], dexamethasone [DXM], and insulin) were from Sigma-Aldrich, Darmstadt, Germany. Isopropanol, 10% formalin, and Oil Red O (ORO) were from Sigma-Aldrich, Darmstadt, Germany. The cell viability assay kit (thiazolyl blue tetrazolium bromide [MTT]) was from Alfa Aesar, Heysham, UK. Dimethyl sulfoxide (DMSO) was from Junsei Chemicals Co Ltd, Japan. Lipopolysaccharide (LPS) from Escherichia coli 0111:B4 and Griess reagent were obtained from Sigma-Aldrich, Darmstadt, Germany.
10
Isolating Primary Cells from Tissue
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