Human fetal lung fibroblasts (MRC5) were purchased from ATCC and umbilical vein cells (HUVECs) were either freshly isolated from donors or purchased from Clonetics, Lonza respectively. The MRC5 were grown in minimum essential medium (MEM, Invitrogen) supplemented with glutamine, 10% fetal bovine serum (FBS) and standard Penicillin and Streptomycin (PS). The HUVEC were cultured in EBM-2 endothelial basal medium supplemented with the EGM-2 Single Quots (Clonetics, Lonza). NIH/3T3 mouse embryonic fibroblast cells were purchased from ATCC and they were cultured in Dulbecco’s Modified Eagle’s medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (FBS) and standard Penicillin and Streptomycin (PS). MycoAlert mycoplasma detection kit (Clonetics, Lonza) was used to verify that cells were mycoplasma-free. The HCMV clinical strain VR1814 (a kind gift from Dr Giuseppe Gerna, University of Pavia, Italy), the HSV-1 (a kind offer from Prof. Maria Masucci’s lab, Karolinska Institute, Sweden), and the MCMV Smith strain (kindly provided by Dr. Frank Stassen, University of Maastricht, Netherlands) were used for the study. The VR1814 virus was propagated in HUVECs and titrated [60 (link)] in MRC5 fibroblasts (ATCC), and frozen at -80°C.
Mycoalert mycoplasma detection kit
Manufactured by Cambrex
Sourced in United States
The MycoAlert Mycoplasma Detection Kit is a biochemical test kit designed to detect the presence of mycoplasma contamination in cell culture samples. The kit utilizes a bioluminescent assay to quantify the activity of mycoplasma-specific enzymes, providing a rapid and sensitive method for mycoplasma detection.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640, by Thermo Fisher Scientific (4 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Huvecs, by Cambrex (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Minimum essential medium (mem), by Thermo Fisher Scientific (1 mentions)
Huvecs, by American Type Culture Collection (1 mentions)
Mrc 5, by Thermo Fisher Scientific (1 mentions)
Human umbilical vein endothelial cells (huvec), by Cambrex (1 mentions)
Egm 2 singlequots, by Cambrex (1 mentions)
Mrc 5 fibroblasts, by American Type Culture Collection (1 mentions)
Mrc 5, by American Type Culture Collection (1 mentions)
Plasmocin, by InvivoGen (1 mentions)
Penicillin, by InvivoGen (1 mentions)
Streptomycin, by InvivoGen (1 mentions)
Sw872, by American Type Culture Collection (1 mentions)
Sw982, by American Type Culture Collection (1 mentions)
A5955, by Merck Group (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
13 protocols using mycoalert mycoplasma detection kit
1
Cultivation of Human Cell Lines and Viral Propagation
2
Standardized Propagation of Human Cytomegalovirus
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The viral stock was a plaque-purified clinical isolate, VR 1814, from cervical secretions (a kind gift from Dr. Giuseppe Gerna, University of Pavia, Italy). The virus was propagated in human umbilical vein cells (HUVECs) [Clonetics, Lonza; or isolated from donors as described [25 (link)], titrated in MRC-5 fibroblasts (ATCC), aliquoted, and frozen at −80°C as described [6 (link)]. Alternatively, the virus was further passaged once in MRC-5 cells, which was subjected to a freeze-thaw cycle; the cellular materials were clarified and ultracentrifuged at 28,000 rpm for 1h at 4°C (Beckman, Optima L-90K Ultracentrifugation). The viral pellet was resuspended in 0.1 M sucrose-phosphate buffer to increase the viral titer as described [26 (link)]. The GBM cell lines U-373 MG (Uppsala), U-251 MG, and U-343 MGa (a kind gift from Dr. M. Nister, Karolinska Institutet, Stockholm, Sweden) were used for in vitro experiments. The growth medium for HUVECs or MRC-5 was as described before [6 (link)]. All GBM cell lines were grown in RPMI supplemented with 10% fetal bovine serum, 100 U/ml of penicillin, 100 μg/ml streptomycin, and 5 μg/ml plasmocin (InvivoGen). All cells were maintained in a humidified 5% CO2 incubator and verified as mycoplasma-free with the MycoAlert mycoplasma detection kit (Clonetics, Lonza).
3
Mycoplasma Detection in Viral Vectors
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Mycoplasma detection was performed on the last harvest of viral vector stocks derived from the 293Vec and the PG13 packaging cell lines in the test runs performed with the iCELLis Nano 200 mL C1 bioreactors using the MycoAlert Mycoplasma Detection Kit, following manufacturer’s recommendations (Cambrex).
4
Culture of Liposarcoma Cell Lines
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The human liposarcoma cell lines SW872 (an undifferentiated liposarcoma, ATCC catalog number: HTB-92) and SW982 (another undifferentiated liposarcoma as evaluated by histopathology, ATCC catalog number: HTB-93) were purchased from the ATCC (Rockville, MD). These cell lines were cultured in RPMI 1640 (Invitrogen, Carlsbad, CA) supplemented with 10% FBS, 100 units/ml penicillin and 100 μg/ml streptomycin (Invitrogen). Cells were incubated at 37°C in a 5% CO2-95% air atmosphere and passaged when near confluent monolayers were achieved using Trypsin-EDTA solution. Cells were free of Mycoplasma contamination, as tested by the MycoAlert Mycoplasma Detection Kit from Cambrex (Rockland, ME).
5
Mycoplasma Detection in Viral Vectors
6
Comprehensive Cell Line Characterization
7
Establishing Multidrug Resistant Cell Lines
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The multidrug resistant U-2OSMR (established by selection against doxorubicin) and U-2OSTR (established by selection against taxol/paclitaxel) was established in our laboratory as previously reported [20 (link), 23 (link)]. The multidrug resistant KHOSR2 (established by selection with doxorubicin) cell line was kindly provided by Dr. Efstathios Gonos (Institute of Biological Research and Biotechnology, Athens, Greece) [29 (link)]. These cell lines were cultured in RPMI 1640 (Invitrogen, CA) supplemented with 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin (Invitrogen). Cells were incubated at 37°C in 5% CO2-95% air atmosphere and passaged when near-confluent monolayers were achieved using trypsin-EDTA solution. Drug resistant cell lines were periodically cultured in the respective drug to confirm their drug resistance characteristics. Cells were free from Mycoplasma contamination as tested using the MycoAlert Mycoplasma Detection Kit from Cambrex (East Rutherford, NJ).
8
Characterization of Osteosarcoma Cell Lines
9
Ovarian Cancer Cell Line Cultivation
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The human ovarian cancer cell lines SKOV3 (ATCC® HTB-77™) and Caov-3 (ATCC® HTB-75™) were purchased from the American Type Culture Collection (Rockville, MD, USA). A2780 (ECACC 93112519) was obtained from the European Collection of Authenticated Cell Cultures. Patricia Donahoe (Massachusetts General Hospital, Boston, MA, USA) provided the human IGROV-1, OVCAR5, and OVCAR8 ovarian cancer cell lines, which have been authenticated and are free of mycoplasma contamination as verified by the MycoAlert Mycoplasma Detection Kit from Cambrex (Rockland, ME, USA). All these cell lines were maintained in RPMI 1640 (GE Healthcare Life Sciences, Logan, UT, USA) medium supplemented with 10% FBS (MilliporeSigma, Burlington, MA, USA) and 1% penicillin/streptomycin (Thermo Fisher Scientific, Waltham, MA, USA) in a humidified incubator containing 5% CO2 at 37°C. The cells were resuspended with 0.05% trypsin-EDTA (Life Technologies Corporation, Grand Island, NY, USA) before subculturing.
10
Establishment and characterization of cell lines
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After obtaining written informed consent, cell lines were established in our laboratory. The MTC-SK cell line (1 (link)) was derived from lymph node metastasis of a 51-year-old central European woman. MTC-SK cells grow as multicellular spheroids in suspension and were maintained in Ham's F12 medium (Biowhittaker, Verviers, Belgium), supplemented with 10% fetal bovine serum (FBS; PAA Laboratories GmbH, Pasching, Austria). HF-SAR fibroblasts (22 (link)) were established from normal skin of a 3-year old male child, cultured in Eagle's minimal essential medium (PAA Laboratories GmbH), supplemented with 10% FBS, in which the cells grew adherently. The cell lines were incubated without antibiotics at 37°C, 5% CO2 and 95% humidity and were Mycoplasma-free, confirmed with a MycoAlert®Mycoplasma Detection kit (Cambrex Bioscience, Rockland, ME, USA) and Hoechst 33258 (Sigma-Aldrich, Vienna, Austria). Hoechst stain (50 μl) was added to 50 μl cell suspension at a density of 2×105 (MTC-SK) and 1×105 (HF-SAR) cells/ml and incubated at room temperature for 5 min. The study was approved by the ethics committee of the Medical University of Graz, Austria (18–182 ex 06/07).
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