All cells were tested for mycoplasma by the luminometric MycoAlert Plus mycoplasma detection kit (Lonza Inc., Rockland, ME, USA) according to the manufacturer’s recommendations. Moreover, authentication of MDA-MB-231 and MDA-hybrid populations was performed by short tandem repeat (STR) fragment analysis using the GenomeLab human STR primer set (Beckman Coulter Inc., Fullerton, CA, USA). The STR pattern of MDA-MB-231 cell line demonstrated similar results according to the STR database provided by the ATCC, Manassas, VA, USA.
Genomelab human str primer set
Manufactured by Beckman Coulter
Sourced in United States
The GenomeLab human STR primer set is a laboratory product designed for the amplification of short tandem repeat (STR) regions of the human genome. It is intended for use in genetic analysis and profiling applications.
Automatically generated - may contain errors
Lab products found in correlation
Mycoalert plus mycoplasma detection kit, by Lonza (3 mentions)
Ceq8800 sequencer, by Beckman Coulter (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
High pure pcr template preparation kit, by Roche (1 mentions)
Hydrocortisone, by Tokyo Chemical Industry (1 mentions)
Human insulin, by Merck Group (1 mentions)
Mcf 10a, by Merck Group (1 mentions)
Mcf 7, by Nissui Pharmaceutical (1 mentions)
Mcf 10a, by American Type Culture Collection (1 mentions)
Human egf, by Thermo Fisher Scientific (1 mentions)
Mcf 7, by American Type Culture Collection (1 mentions)
Hek293t, by American Type Culture Collection (1 mentions)
Bt549, by American Type Culture Collection (1 mentions)
Dmem f12, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Biowest (1 mentions)
Dulbecco modified eagle medium (dmem), by Nissui Pharmaceutical (1 mentions)
Rpmi 1640, by Nissui Pharmaceutical (1 mentions)
Hek 293t, by Nissui Pharmaceutical (1 mentions)
8 protocols using genomelab human str primer set
1
Mycoplasma and Cell Line Authentication
2
Mycoplasma and STR Authentication of Cell Lines
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All cells were tested for mycoplasma by the luminometric MycoAlert Plus mycoplasma detection kit (Lonza Inc., Rockland, ME, USA) according to the manufacturer’s recommendations. Cell line authentication was performed by short tandem repeat (STR) fragment analysis using the GenomeLab human STR primer set (Beckman Coulter Inc., Fullerton, CA, USA). The STR pattern of SK-OV-3 ovarian cancer cells was confirmed in previous work [30 (link)] according to the STR database provided by the ATCC, Manassas, VA, USA.
3
Authentication of Pancreatic Cancer Cell Lines
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The human pancreatic adenocarcinoma cell lines Panc1, PaTu-8988T, BxPC3 and IMIMPC2 were used in this study. Panc1 and BxPC3cells were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Germany). PaTu-8988T cells were kindly provided by H. P. Elsässer (Cytobiology and Cytopathology Institute, Philipps University, Marburg, Germany). IMIM-PC2 cells were kindly provided by F.X. Real (CNIO, Madrid, Spain). These cancer cell lines were maintained in Dulbecco’s modified minimal essential medium (GIBCO, USA) supplemented with 10% FCS (GIBCO, USA) and cultured at 37°C/ 5% CO2.
Cancer cell line identities were verified using the GenomeLab Human STR Primer Set (Beckman Coulter, Krefeld, Germany) on a CEQ8800 sequencer (Beckman Coulter) according to the manufacturer’s protocol. STR data were submitted to on-line verification tool of DSMZ (www.dsmz.de ) to confirm identity of human cell lines.
Surgically resected PDAC and chronic pancreatitis tissues samples were procured from the surgery department of the University of Heidelberg. Samples of normal pancreatic tissue were obtained from healthy donors. Informed consent in writing was obtained from all patients prior to using tissue samples. The study was approved by the ethics committee at the University of Heidelberg, Germany.
Cancer cell line identities were verified using the GenomeLab Human STR Primer Set (Beckman Coulter, Krefeld, Germany) on a CEQ8800 sequencer (Beckman Coulter) according to the manufacturer’s protocol. STR data were submitted to on-line verification tool of DSMZ (
Surgically resected PDAC and chronic pancreatitis tissues samples were procured from the surgery department of the University of Heidelberg. Samples of normal pancreatic tissue were obtained from healthy donors. Informed consent in writing was obtained from all patients prior to using tissue samples. The study was approved by the ethics committee at the University of Heidelberg, Germany.
4
Characterization of Breast Cancer Cell Lines
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Chemicals were obtained from Sigma-Aldrich, Poole, Dorset, UK, unless otherwise stated. All cell lines were recently obtained from the ATCC and are part of a collection of breast cancer cells held at the University of Westminster and University College London. The cells have been confirmed mycoplasma free. SKBR-3, MCF-7, ZR75–1 cells were subjected to short tandem repeat (STR) typing performed in-house using the Genome Lab Human STR Primer Set (Beckman Coulter, High Wycombe, UK). DNA samples were prepared using the high-pure PCR template preparation kit (Roche, Welwyn Garden City, UK). Fragment analysis was performed on a Beckman Coulter CEQ8800 Genetic Analyser and the STR genotype profile of each cell line was analysed using an online tool provided by the DMSZ collection of cell cultures https://www.dsmz.de/ .
5
Mycoplasma and Cell Line Authentication Protocol
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All cells were tested for mycoplasma by the luminometric MycoAlert Plus mycoplasma detection kit (Lonza Inc., Rockland, ME, USA) according to the manufacturer's recommendations. Moreover, authentication of SCCOHT-1, BIN-67, NIH:OVCAR-3, and SK-OV-3 cell lines was performed by short tandem repeat (STR) fragment analysis using the GenomeLab human STR primer set (Beckman Coulter Inc., Fullerton, CA, USA). The fragment analysis for the NIH:OVCAR-3 and SK-OV-3 cell lines demonstrated a similar STR pattern according to the STR database provided by the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany).
Whereas no STR patterns are available for SCCOHT-1 and BIN-67 cells to date, repeated STR fragment analyses were performed for these two cell populations. Data from different SCCOHT-1 culture periods (n = 3) confirmed similar patterns and these STR pattern were presented inSuppl. Fig. S6 .
Whereas no STR patterns are available for SCCOHT-1 and BIN-67 cells to date, repeated STR fragment analyses were performed for these two cell populations. Data from different SCCOHT-1 culture periods (n = 3) confirmed similar patterns and these STR pattern were presented in
6
PDAC Cell Line Characterization and PPSC Isolation
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Capan1 and SW1990 cells were cultured in DMEM, BxPC3, and SU86.86 cells in RPMI; and Panc0403 cells in RPMI supplemented with 10 mm HEPES buffer, 1 mm sodium pyruvate, and 0.2 U/ml insulin from bovine pancreas. Media contained 10% fetal bovine serum and 2 mm l ‐glutamine. The absence of Mycoplasma was regularly confirmed using PCR. DNA fingerprinting of the PDAC cell lines (Capan1, BxPC3, and Panc0403) was performed using the GenomeLab Human STR Primer Set (Beckman‐Coulter Inc, Brea, CA, USA), and the results were verified against the COSMIC cell line database, Wellcome Trust Sanger Institute. Primary pancreatic stellate cells (PPSCs) were obtained using normal pancreatic tissue from two patients undergoing pancreatic resection at University Hospital Southampton (UHS). Appropriate ethical approval and patient consent were in place (REC No 10/H0502/72).
7
Maintenance of Human Cell Lines
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HeLa, MCF7, HEK-293T, MCF10A, and BT-549 cells were purchased from ATCC. HeLa, MCF7, and HEK-293T cells were maintained in DMEM (Nissui Pharmaceutical) supplemented with 8% FBS (Biowest) in an atmosphere with 5% CO2. MCF10A cells were maintained in DMEM/F-12 (Sigma-Aldrich) supplemented with 5% FBS, 20 ng/mL human EGF (PeproTech), 10 μg/mL human insulin, and 0.5 μg/mL hydrocortisone (Tokyo Chemical Industry) in an atmosphere with 5% CO2. BT-549 cells were maintained in RPMI1640 (Nissui pharmaceutical) supplemented with 8% FBS and 10 μg/mL human insulin (Sigma-Aldrich) in an atmosphere with 5% CO2. Cell line identities were verified using the GenomeLab Human STR Primer set (Beckman Coulter). Cells were routinely stained by Hoechst 33342 and no mycoplasma contamination was suspected. Cells were used for experiments within 20 passages from obtaining.
8
Identifying Gefitinib-Resistant Lung Cancer Cells
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A total of 2×105 cells were plated in ten 10-cm plates and allowed to adhere for 24 hr. Cells were then treated with gefitinib at a concentration of 1 µM, which is 30–50 times the established IC50 values, and incubated under normoxia (21% O2) or hypoxia (1% O2). Media was replaced with Fresh media containing gefitinib every 3 days. Viable cells that remained attached on the dish at day 7 under normoxic or hypoxic conditions were considered to be gefitinib-resistant persisters (GRPs) or hypoxic gefitinib-resistant persisters (hypoxic GRPs), respectively, and were collected for analysis. The genetic identity of GRPs with the parental cells was confirmed using the GenomeLab Human STR Primer set from Beckman Coulter (Fullerton, CA, USA), according to the manufacturer’s instructions.
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