Powerplex 16 system

Manufactured by Promega

Sourced in United States, United Kingdom, Germany, Spain, Japan

The PowerPlex 16 System is a forensic short tandem repeat (STR) multiplex assay used for human identity testing. The system amplifies 16 genetic loci, including the 13 CODIS core loci and Amelogenin, in a single PCR reaction. The resulting amplified fragments can be detected and analyzed using capillary electrophoresis instrumentation.

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134 protocols using powerplex 16 system

NHL Cell Line Authentication and Maintenance

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The NHL cell lines WSU‐DLCL2 (DSMZ Cat# ACC‐575, RRID:CVCL_1902), BJAB (DSMZ Cat# ACC‐757, RRID:CVCL_5711), and BJAB.PD.cyCD79b.E3 were obtained from the Genentech (RRID:SCR_003997) cell line repository and maintained in RPMI‐1640 supplemented with 10% FBS (Sigma) and 2‐mM l‐glutamine. Each cell line was authenticated by short tandem repeat profiling using the Promega PowerPlex 16 System (Promega Corporation, Madison, WI [RRID:SCR_006724]) and compared with external short tandem repeat profiles of cell lines to determine cell line ancestry.

Li D., Lee D., Dere R.C., Zheng B., Yu S., Fuh F.K., Kozak K.R., Chung S., Bumbaca Yadav D., Nazzal D., Danilenko D., Go M.A., Williams M., Polson A.G., Poon K.A, & Prabhu S. (2019). Evaluation and use of an anti‐cynomolgus monkey CD79b surrogate antibody–drug conjugate to enable clinical development of polatuzumab vedotin. British Journal of Pharmacology, 176(19), 3805-3818.

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STR Genotyping of Cell Lines

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DNA was extracted with the High Pure PCR Template Preparation kit (Roche Diagnostics GmbH, Manheim, Alemania) from the cell line (passage 35), the primary tumor and from normal blood lymphocytes of the same patient. Short tandem repeat (STR) genotyping was performed using the Promega Powerplex 16 system (Promega Biotech Ibérica SL, Barcelona, Spain), analyzing fifteen STR loci (Penta E, D18S51, D21S11, TH01, D3S1358, FGA, TPOX, D8S1179, vWA, Penta D, CSF1PO, D16S539, D7S820, D13S317 and D5S818) and Amelogenin by PCR.

Lorenzo-Guerra S.L., Codina-Martínez H., Suárez-Fernández L., Cabal V.N., García-Marín R., Riobello C., Vivanco B., Blanco-Lorenzo V., Sánchez-Fernández P., López F., Llorente J.L, & Hermsen M.A. (2023). Characterization of a Preclinical In Vitro Model Derived from a SMARCA4-Mutated Sinonasal Teratocarcinosarcoma. Cells, 13(1), 81.

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Oxaliplatin-Resistant Neuroblastoma Cell Lines

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The non-MYCN-amplified neuroblastoma cell line SK-N-AS was obtained from ATCC (Manassas, VA, US). The oxaliplatin-resistant SK-N-AS sub-line SK-N-AS^rOXALI⁴⁰⁰⁰ adapted to growth in the presence of oxaliplatin 4000 ng/mL was derived from the resistant cancer cell line (RCCL) collection (www.kent.ac.uk/stms/cmp/RCCL/RCCLabout.html) and had been established by previously described methods [22 (link)]. In addition, we used an SK-N-AS^rOXALI⁴⁰⁰⁰ sub-line that had been cultivated for at least 10 passages in the absence of oxaliplatin (SK-N-AS^rOXALI^4000(-)) as a control. The MYCN-amplified UKF-NB-3 neuroblastoma cell line was established from bone marrow metastases of a stage IV neuroblastoma patient [23 (link)].
All cells were propagated in IMDM supplemented with 10% FBS, 100 IU/ml penicillin and 100 μg/ml streptomycin at 37°C. Cells were routinely tested for mycoplasma contamination. Authentication was performed by short tandem repeat (STR) profiling. DNA was isolated using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany), and the STR analysis was performed using the PowerPlex 16 System (Promega, Mannheim, Germany) according to the manufacturers' protocols.

Saintas E., Abrahams L., Ahmad G.T., Ajakaiye A.O., AlHumaidi A.S., Ashmore-Harris C., Clark I., Dura U.K., Fixmer C.N., Ike-Morris C., Mato Prado M., Mccullough D., Mishra S., Schöler K.M., Timur H., Williamson M.D., Alatsatianos M., Bahsoun B., Blackburn E., Hogwood C.E., Lithgow P.E., Rowe M., Yiangou L., Rothweiler F., Cinatl J j.r., Zehner R., Baines A.J., Garrett M.D., Gourlay C.W., Griffin D.K., Gullick W.J., Hargreaves E., Howard M.J., Lloyd D.R., Rossman J.S., Smales C.M., Tsaousis A.D., von der Haar T., Wass M.N, & Michaelis M. (2017). Acquired resistance to oxaliplatin is not directly associated with increased resistance to DNA damage in SK-N-ASrOXALI4000, a newly established oxaliplatin-resistant sub-line of the neuroblastoma cell line SK-N-AS. PLoS ONE, 12(2), e0172140.

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Cell Line Authentication and Culture

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Cell lines were purchased from the American Type Culture Collection (ATCC, LGC Promochem, Molsheim, France) and authenticated (data not shown) by short tandem repeat profiling in 2018 using the PowerPlex^® 16 System (Promega, Charbonnieres les bains, France; DC6531) [51 (link)]. Cells lines were cultured as previously described [52 (link),53 (link),54 (link)]. Briefly, Hs578t and MDA-MB-157 cells were cultured in D MEM (Thermo Fisher Scientific, Courtaboeuf, France; 31966047) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 µg/mL streptomycin (P/S; Thermo Fisher Scientific, Courtaboeuf, France; 10378016). BT-474 and MDA-MB-436 cells were maintained in RPMI-1640 (Thermo Fisher Scientific, Courtaboeuf, France; 61870044) supplemented with 10% and P/S. MDA-MB-231 cells were cultured in D MEM-F12 (Thermo Fisher Scientific, Courtaboeuf, France; 31331093) supplemented with 10% FBS and P/S. MCF7 cells were cultured in MEM (Sigma-Aldrich, Saint-Quentin Fallavier, France; M2279) containing 10% FBS, 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids (Thermo Fisher Scientific, Courtaboeuf, France; 11140050), 1 mM sodium pyruvate and P/S. We maintained all cell lines at 37 °C in a humidified atmosphere with 5% CO₂.

Zajac O., Leclere R., Nicolas A., Meseure D., Marchiò C., Vincent-Salomon A., Roman-Roman S., Schoumacher M, & Dubois T. (2020). AXL Controls Directed Migration of Mesenchymal Triple-Negative Breast Cancer Cells. Cells, 9(1), 247.

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SCLC Cell Line Authentication

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SCLC cell lines GLC-2 [17] (link) and -36 [18] (link) were provided by Prof. Dr. L.F.M.H. de Leij (University of Groningen, The Netherlands) and SCLC-24H [19] (link) by Dr. H. Lahm (Thoraxklinik Heidelberg, Germany). SCLC cell line H146 was purchased from American Type Culture Collection (Rockville, USA) and SCLC cell line H1184 from DSMZ (Braunschweig, Germany). Cell lines were maintained in DMEM, 10% fetal calf serum (FCS) or in DMEM/Ham'sF12, 20% FCS (cell line H1184). For DNA profiling, 15 different short tandem repeat (STR) systems and the gender specific amelogenin locus were amplified from 1 to 5 ng genomic DNA in a multiplex-PCR using the PowerPlex® 16 System (Promega, Madison, WI, USA), dedicated to forensic DNA profiling, according to manufacturer's instructions. PCR products were quality controlled utilizing a micro fluidic ‘DNA 7500’ array on a 2100 Bioanalyzer (Agilent, Böblingen, Germany). If quality control was passed, allele detection and identification was performed by capillary electrophoresis performed on a 310 Genetic Analyzer (Applied Biosystems, Darmstadt, Germany). Data processing and analysis was done using the GeneMapper software (v3.2) (Applied Biosystems). The DNA profiles of the cell lines are shown in Table 1.

Murmann T., Carrillo-García C., Veit N., Courts C., Glassmann A., Janzen V., Madea B., Reinartz M., Harzen A., Nowak M., Perner S., Winter J, & Probstmeier R. (2014). Staurosporine and Extracellular Matrix Proteins Mediate the Conversion of Small Cell Lung Carcinoma Cells into a Neuron-Like Phenotype. PLoS ONE, 9(2), e86910.

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Genetic Profiling of Donor BMMCs

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PowerPlex 16 System (Promega) was used in conjunction with an Applied Biosystems (Life Technologies) 3130xl Genetic Analyzer. Donor BMMCs were used as the reference baseline.

Zheng G.X., Terry J.M., Belgrader P., Ryvkin P., Bent Z.W., Wilson R., Ziraldo S.B., Wheeler T.D., McDermott G.P., Zhu J., Gregory M.T., Shuga J., Montesclaros L., Underwood J.G., Masquelier D.A., Nishimura S.Y., Schnall-Levin M., Wyatt P.W., Hindson C.M., Bharadwaj R., Wong A., Ness K.D., Beppu L.W., Deeg H.J., McFarland C., Loeb K.R., Valente W.J., Ericson N.G., Stevens E.A., Radich J.P., Mikkelsen T.S., Hindson B.J, & Bielas J.H. (2017). Massively parallel digital transcriptional profiling of single cells. Nature Communications, 8, 14049.

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PDAC Cell Line Characterization and Culture

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Two human PDAC cell line models were included in this study - Paca-44 and BxPC-3. Paca-44 (carrying KRAS G12V and TP53 C176S mutations) was obtained from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany (product. no. ACC 179) and the BxPC-3 cell line (KRAS wild type) was obtained from CLS (Eppelheim, Germany, product no. 305031). Cells were cultured in RPMI 1640 medium with L-Glutamine (300 mg/L), NaHCO₃ (2 g/L), penicillin-streptomycin (penicillin 100 U/mL, streptomycin 100 μg/mL), sodium pyruvate (1 mM), HEPES (15 mM) and fetal bovine serum or heat-inactivated fetal bovine serum (PAN-Biotech, Aidenbach, Germany) at final concentration 10% for Paca-44 or BxPC-3 cells, respectively. The cell lines were cultured at 37°C in an incubator with a 5% CO₂ atmosphere. MycoAlert mycoplasma detection kit (Lonza, Basel, Switzerland) was used for regular mycoplasma contamination tests. The authenticity of the cells was checked by short tandem repeats (STR) DNA profiling analysis. For amplification of STR loci, a PowerPlex 16 System (Promega, Madison, WI, USA) was used according to the technical manual.

Sychra T., Spalenkova A., Balatka S., Vaclavikova R., Seborova K., Ehrlichova M., Truksa J., Sandoval-Acuña C., Nemcova V., Szabo A., Koci K., Tesarova T., Chen L., Ojima I., Oliverius M, & Soucek P. (2024). Third-generation taxanes SB-T-121605 and SB-T-121606 are effective in pancreatic ductal adenocarcinoma. iScience, 27(2), 109044.

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DNA Extraction and STR Profiling from Tumor Tissue

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DNA from tumor tissue and cells was prepared using the QIAamp DNA Mini kit (Qiagen, Hilden, Germany) in accordance with the manufacturer’s protocol. After normalizing the DNA, 1 μl of each sample was amplified using the Power Plex® 16 System (Promega, Madison, WI) in a 10 μl reaction. One μl of the product was mixed with Hi-Di formamide (Applied Biosystems Inc., US) and Internal Lane Standard (ILS600), denatured and fractionated on an ABI 3730 Genetic Analyzer (Applied Biosystems Inc.). The resulting data were processed and evaluated using ABI Genemapper 4.0⁴¹.

Heitzer E., Groenewoud A., Meditz K., Lohberger B., Liegl-Atzwanger B., Prokesch A., Kashofer K., Behrens D., Haybaeck J., Kolb-Lenz D., Koefeler H., Riedl S., Schaider H., Fischer C., Snaar-Jagalska B.E., de’Jong D., Szuhai K., Zweytick D, & Rinner B. (2019). Human melanoma brain metastases cell line MUG-Mel1, isolated clones and their detailed characterization. Scientific Reports, 9, 4096.

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Lung Cancer Cell Culture Protocols

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Non-small cell lung cancer cell lines A549 (KRAS G12S) (Georgetown Lombardi Comprehensive Cancer Center Tissue Culture Shared Resource, TCSR) and H1299 (NRAS Q61K) (ATCC) were cultured in RPMI 1640 medium supplemented with heat-inactivated 10% fetal bovine serum (FBS) and 100 U/mL penicillin and 100 μg/mL streptomycin (P/S) (Life Technologies, Grand Island, NY). LM2–4175 cells, lung metastatic derivative of MD-MB-231 breast cancer cells (KRAS, G13D; BRAF, G464V; TP53, R280K), were a kind gift from Dr. Joan Massagué. Breast (MDA-MB-231 and LM2–4175), melanoma (MDA-MB-435), prostate (PC-3, TP53, K139fs31-del frame shift and C4–2B, TP53 wt) and pancreatic cancer cells (PANC-1, KRAS, G12D; TP53, R273H) (TCSR), and HEK293T cells (ATCC) were grown in Dulbecco’s modified essential medium (DMEM) with Glutamax and high glucose (Life Technologies) supplemented with heat-inactivated 10% FBS and P/S. Various cell lines were authenticated at TCSR using PowerPlex® 16 System (Promega). Mycoplasma testing at TCSR was done using the MycoAlert kit (Lonza). All cell lines were passaged and used in the laboratory within six months of receipt from TCSR or ATCC. All cultures were maintained in a humidified incubator at 37^oC with 5% CO₂.

Day T.F., Kallakury B.V., Ross J.S., Voronel O., Vaidya S., Sheehan C.E, & Kasid U.N. (2019). Dual Targeting of EGFR and IGF1R in the TNFAIP8 Knockdown Non-small Cell Lung Cancer Cells. Molecular cancer research : MCR, 17(5), 1207-1219.

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Cell Line Authentication and Western Blotting

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Cell lines used for Western blotting were obtained from the American Type Culture Collection (ATCC), National Cancer Institute Division of Cancer Treatment and Diagnosis Tumor Repository (Bethesda, MD); or Japan Health Sciences Foundation (Tokyo, Japan) and were authenticated. For authentication, short tandem repeat (STR) profiles were determined for each line using the Promega PowerPlex 16 System. This was performed once and compared with external STR profiles of cell lines (when available) to determine cell line ancestry. Sixteen loci (fifteen STR loci and Amelogenin for gender identification) were analyzed, including D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, AMEL, vWA, D8S1179, and TPOX. NSCLC cell lines were lysed in T-PER tissue extraction reagent (Thermo Scientific) supplemented with protease and phosphatase inhibitors. Proteins resolved by SDS–PAGE were electrophoretically transferred to polyvinylidene difluoride membrane and Western blots were carried out using standard techniques. The primary antibodies used in this study were: MET clone SP44 (Ventana Medical Systems), MET clone L41G3 (Cell Signaling Technology), MET clone 5D5 (HB-11895; ATCC), and actin (cat. no. sc-8432; Santa Cruz Biotechnology). A polyclonal antibody raised against MST1R was a kind gift from Dr Amitabha Chaudhuri (Genentech, Inc.).

Koeppen H., Yu W., Zha J., Pandita A., Penuel E., Rangell L., Raja R., Mohan S., Patel R., Desai R., Fu L., Do A., Parab V., Xia X., Januario T., Louie S.G., Filvaroff E., Shames D.S., Wistuba I., Lipkind M., Huang J., Lazarov M., Ramakrishnan V., Amler L., Phan S.C., Patel P., Peterson A, & Yauch R.L. (2014). Biomarker Analyses from a Placebo-Controlled Phase II Study Evaluating Erlotinib ± Onartuzumab in Advanced Non–Small Cell Lung Cancer: MET Expression Levels Are Predictive of Patient Benefit. Clinical cancer research : an official journal of the American Association for Cancer Research, 20(17), 4488-4498.

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