The DDLPS cell lines NRH-LS1, established from a patient-derived xenograft as previously described [3 (link)] and LPS510 and LPS853, kindly provided by Dr. Jonathan Fletcher, were cultured in RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% FBS (Sigma-Aldrich), 1% l -alanyl-l -glutamine (Sigma-Aldrich) and 1% penicillin-streptomycin (Sigma-Aldrich) and grown at 37 °C, 5% CO2. Short tandem repeat DNA profiling was performed on all cell lines to confirm identity. Cells were negative for mycoplasma using the VenorGeM Mycoplasma Detection Kit (Minerva Biolabs, Berlin, Germany).
Venor gem mycoplasma detection kit
Manufactured by Minerva Biolabs
Sourced in Germany
The Venor GeM Mycoplasma Detection Kit is a molecular detection kit designed to identify the presence of mycoplasma contamination in cell cultures. The kit utilizes a polymerase chain reaction (PCR) assay to detect a highly conserved region of the 16S rRNA gene, which is common to a wide range of mycoplasma species.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (10 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (8 mentions)
Penicillin streptomycin, by Merck Group (5 mentions)
Fetal bovine serum (fbs), by Merck Group (4 mentions)
Glutamax, by Thermo Fisher Scientific (4 mentions)
L alanyl l glutamine, by Merck Group (3 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (3 mentions)
Mda mb 231, by American Type Culture Collection (3 mentions)
Aligner, by CodonCode (3 mentions)
Rpmi 1640 medium, by Merck Group (2 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions)
Hepes, by Merck Group (2 mentions)
150 cm2 tissue culture flasks, by Eppendorf (2 mentions)
4 2 hydroxyethyl 1 piperazineethanesulfonic acid, by Merck Group (2 mentions)
Dmem ham s 12 1 1, by Harvard Bioscience (2 mentions)
Mcf 7, by American Type Culture Collection (2 mentions)
L glutamine, by Merck Group (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Ht1376, by American Type Culture Collection (1 mentions)
Coating matrix kit, by Thermo Fisher Scientific (1 mentions)
52 protocols using venor gem mycoplasma detection kit
1
Characterization of DDLPS cell lines
2
Urothelial Cancer Cell Line Characterization
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The chemotherapeutics, gemcitabine and cisplatin, were obtained ready-to-use from the in-house pharmacy of the RWTH Aachen University Hospital. The urothelial cancer cell line HT1376 (basal-type) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). SCaBER, a basal-type bladder cancer cell line with squamous characteristics, was a kind gift of Prof. Wolfgang Schulz/Dr. Michèle Hoffmann (Düsseldorf University Hospital, Düsseldorf, Germany). All cell lines were cultured using the DMEM (Dulbecco’s Modified Eagle’s Medium) (Sigma-Aldrich, Deisenhofen, Germany) supplemented with 10% FCS (Gibco Laboratories, Berlin, Germany), and successfully underwent an identity check (Multiplexion GmbH, Immenstadt, Germany) prior to the experiments. All cells and clones were regularly tested for mycoplasma infection using the PCR-based Venor® GeM Mycoplasma Detection Kit (Minerva Biolabs, Berlin, Germany).
3
Cultivation and Characterization of CIEB Cells
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Cells were maintained in high glucose (4.5 g/L) Gibco® D-MEM medium (Invitrogen, Thermo Scientific, Vienna, Austria). Media was supplemented with 10% fetal bovine serum (Life Technologies, Thermo Scientific, Vienna, Austria), 16 mM HEPES (Sigma Aldrich, Vienna, Austria), Gibco® 2.5 mM GlutaMAX™, and penicillin/streptomycin (100 units/mL; 100 µg/mL; Sigma Aldrich)). CIEB were cultivated at 37 °C in a 5% CO2 atmosphere (Galaxy 48 S, New Brunswick, Eppendorf, Hamburg, Austria). At first passage, coated (Coating Matrix Kit, Life Technologies) 25-flasks (Star Lab, Hamburg, Germany) were used. Thereafter, cells were cultivated in uncoated 75-flasks (Star Lab). CIEB were subcultured two to three times a week upon reaching 80% confluence. Cells were used until passage 24 and regularly confirmed to be free of mycoplasma contamination via PCR (Venor® GeM Mycoplasma Detection Kit; Minerva Biolabs, Berlin, Germany).
4
Establishment and Characterization of Sarcoma Cell Lines
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All cell lines were established in our laboratories (Solid Tumor Genetics Laboratory of Nice University Hospital, Nice, France and Institut Bergonié, Bordeaux, France) from a primary retroperitoneal WDLPS (93T449), a primary periscapular DDLPS (IB111), a primary paratesticular DDLPS (IB115), and a soft-tissue leiomyosarcoma (LMS) (IB136) [51 (link)]. All cell lines were genomically characterized by array-CGH and tested for mycoplasma using the VenorGeM Mycoplasma Detection Kit (Minerva Biolabs, Berlin, Germany). The cells were cultured in RPMI-1640 medium containing GlutaMAX (Life Technologies, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Life Technologies, Waltham, MA, USA) and 1% penicillin-streptomycin (Sigma-Aldrich, St. Louis, MO, USA) and grown at 37 °C, with 5% CO2.
5
HEK293T Cell Culture and Mycoplasma Testing
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In this study, the HEK293T cell line, obtained from the German collection of microorganisms and cell cultures (DSMZ), was used. Cells were routinely monitored for mycoplasma contamination using the Venor GeM Mycoplasma Detection Kit (Minerva Biolabs, Germany) and were negative. Unless otherwise stated, the cells were cultivated in DMEM containing 10% FCS (full medium) at 37 °C in a water-saturated atmosphere containing 5% CO2.
6
Osteosarcoma Cell Line Authentication
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The human cell line hFOB1.19, human osteosarcoma cell lines MG-63, u2os, SJSA-1, HOS and 143B were purchased from FuHeng Cell Center (Shanghai, China). The OS cell lines were authenticated at the by the ShangHai Biowing Applied Biotechnology Co. Ltd., performing a STR profiling analysis, as described by Capes-Davis and according to the ANSI Standard (ASN-0002) set forth by the ATCC Standards Development Organization. Mycoplasma testing was performed using the Venor GeM Mycoplasma Detection Kit (Minerva Biolabs, Berlin, Germany). Cells were cultured as described in detail in the Additional file 1 : Materials section.
7
Cell Line Maintenance and Mycoplasma Testing
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The original cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and routinely tested for mycoplasma contamination using VenorGem Mycoplasma Detection Kit (Minerva Biolabs GmbH; Berlin Germany). NIH3T3 mouse fibroblasts, EMT6 mouse breast cancer cell lines and HEK293T cells were maintained in complete medium composed of Dulbecco's Modified Eagle's Medium (DMEM; PAA Laboratories GmbH, Pasching, Austria) supplemented with 10% fetal bovine serum (PAA Laboratories GmbH, Pasching, Austria) and 80 μg/mL gentamycin (KRK, Novo Mesto, Slovenia). Cells were grown using standard culture conditions (37°C, humidified atmosphere containing 5% CO2).
8
Culturing Endothelial and Osteosarcoma Cells
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The human umbilical vein endothelial cell line HUVEC (RRID: CVCL_2959, female), mouse endothelial cell line EOMA (RRID: CVCL_3507), and human osteosarcoma cell line 143B (RRID: CVCL_2270, female) were purchased from American Type Culture Collection (ATCC). HUVECs were maintained in endothelial cell medium (ECM, ScienCell, USA) supplemented with 5% fetal bovine serum (FBS, ScienCell, USA) and endothelial cell growth supplement (ECGS, ScienCell, USA). EOMA cells were maintained in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 10% FBS (Gibco, Grand Island, NY, USA). 143B cells were maintained in Eagle’s minimum essential medium (EMEM) supplemented with 10% FBS. Primary hepatocytes were isolated from mice as described below and cultured in DMEM supplemented with 10% FBS. All cells were incubated at 5% CO2 at 37 °C. Mycoplasma infection was ruled out in all cell lines using the Venor GeM Mycoplasma Detection Kit (Minerva Biolabs, Berlin, Germany).
9
CHO-K1 cells expressing hD2long Receptor
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CHO-K1 hD2longR cells70 (link) were a kind gift from Dr. Harald Hübner (Department of Chemistry and Pharmacy, Friedrich-Alexander-University, Erlangen). These cells were cultured in DMEM/F-12 supplemented with 10% FCS and 600 μg/mL G418 at 37 °C in a water-saturated atmosphere containing 5% CO2. Cells were routinely tested for mycoplasma contamination using the Venor GeM Mycoplasma Detection Kit (Minerva Biolabs, Germany).
10
Mycoplasma-free Breast Cancer Cell Lines
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The human breast cancer cell lines SKBR3 and Hs578T were originally obtained from the American Type Culture Collection (Rockville, MD, US) and cultured under recommended conditions. Cell lines were regularly tested for mycoplasma infection using the PCR-based Venor® GeM Mycoplasma Detection Kit (Minerva Biolabs, Berlin, Germany).
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