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Ampfistr identifier kit

Manufactured by Thermo Fisher Scientific
Sourced in United States

The AmpFISTR Identifier kit is a genetic analysis tool designed for forensic and human identification applications. It enables the amplification and detection of specific DNA markers to generate a genetic profile for identification purposes. The kit provides the necessary reagents and protocols to perform the analysis.

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23 protocols using ampfistr identifier kit

1

Sensitizing Cervical Cancer Cells

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The human cervical cancer cell lines HeLa (HPV 18-positive) and CaSki (HPV16-positive) were obtained from the ATCC (Manassas, VA). Cells were routinely tested for mycoplasma and characterized by short tandem report profiling (AmpFISTR identifier kit, Applied Biosystems, Foster, CA, cat. 4322288). HeLa and CaSki cells were transfected with siRNAs using DharmaFECT transfection reagent (Thermo Scientific, USA) according to the manufacturer's protocol. The HPV 16 and 18 siRNA sequences are listed in Table S1. Cisplatin (P4394) and paclitaxel (T7191) were obtained from Sigma-Aldrich, USA. For combination therapy, cells were exposed to E6/E7 siRNA along with chemotherapeutic agents.
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2

Isolating and Authenticating GBM Neurospheres

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The GIC lines were established by isolating neurosphere-forming cells from fresh surgical specimens of human GBM tissue between the years of 2005 through 2008, as described previously (18 (link)). Cells were authenticated by testing short tandem repeats (STR) using the Applied Biosystems AmpFISTR Identifier kit (Foster City, CA). The last authentication testing was done in March 2014. This study was approved by the institutional review board of The University of Texas MD Anderson Cancer Center (Houston, Texas). These GBM neurospheres were cultured in DMEM/F12 medium containing B27 supplement (Invitrogen, Carlsbad, CA), basic fibroblast growth factor, and epidermal growth factor (20 ng/ml each). The PI3K/mTOR dual inhibitor BEZ235 was from Selleck (Houston, Texas), DS-7423 was provided by Daiichi Sankyo Co., Ltd. (Tokyo, Japan), and PD-0325901 was from Selleck (Houston, Texas). For in vitro use, all inhibitors were dissolved in dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO).
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3

Patient-Derived Glioma Sphere Culture

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Patient-derived glioma sphere-forming cell (GSC) lines with varying EGFR and p53 status were used in this study. The GSCs were established by isolating neurosphere-forming cells from fresh surgical specimens of GBM between 2005 and 2008, as described previously.28 (link) Cells were authenticated by testing short tandem repeats using the Applied Biosystems AmpFISTR Identifier kit (Foster City, CA). The most recent authentication was performed on July 31, 2017. This study was approved by the Institutional Review Board of The University of Texas MD Anderson Cancer Center. The GSC lines were cultured in DMEM/F12 medium containing B27 supplement (Invitrogen, Carlsbad, CA), basic fibroblast growth factor (20 ng/mL), and epidermal growth factor (20 ng/mL). Erlotinib and nedisertib were purchased from Selleckchem (Houston, TX).
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4

Culturing Human Neuroblastoma Cell Lines

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Human-derived neuroblastoma cell lines (Table 1) were obtained from the Children’s Hospital of Philadelphia cell line bank, the Children’s Oncology Group, or ATCC (American Type Culture Collection) and were cultured in RPMI-1640 medium (Invitrogen) containing 10% FBS (Hyclone), 2 mM L-Glutamine and 1% streptomycin/penicillin at 37°C under 5% CO2. Cells tested negative for mycoplasma contamination and were authenticated by a short tandem repeat profile using the AmpFISTR Identifier kit (Applied Biosystems).
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5

Co-culture of AML Cells with MSCs

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The human AML cell lines OCI-AML3, MOLM13, and MV4-11 were kindly provided by Dr. Marina Konopleva (MD Anderson Cancer Center). The identity of all cell lines was validated by short tandem repeat DNA fingerprinting using the AmpFISTR identifier kit according to manufacturer’s instructions (Applied Biosystems). All cell lines were maintained at 37 °C with 5% CO2 in a humidified incubator in a culture medium consisting of RPMI-1640 (Sigma Aldrich, St. Louis, MO, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Atlanta Biologicals, Flowery Branch, GA, USA), penicillin, and streptomycin.
Co-culture experiments were performed by incubating AML cells with a feeder layer of human mesenchymal stem cells (MSC) derived from normal bone marrow samples in accordance with institutional guidelines. MSC cultures were plated for 24 h at 37 °C with 5% CO2 in a culture medium consisting of 20% FBS (Fisher Scientific, Hanover Park, IL, USA), α-MEM (Corning, Manassas, VA, USA) and 4% Human Platelet Lysate (EMD Millipore Corp, Billerica, MA, USA). Thereafter, the culture medium was removed and AML cells (OCI/AML3) were seeded on top of the MSC layer in a ratio of 4:1 (OCI/AML3:MSC) in RPMI-1640 and 10% FBS. Co-cultured cells were kept for 24 h at 37 °C with 5% CO2 before treatment (see below).
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6

Culturing Human Neuroblastoma Cell Lines

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Human-derived neuroblastoma cell lines (Table 1) were obtained from the Children's Hospital of Philadelphia cell line bank, the Children's Oncology Group, or ATCC and were cultured in RPMI-1640 medium (Invitrogen) containing 10% FBS (Hyclone), 2 mmol/L l-Glutamine, and 1% streptomycin/penicillin at 37°C under 5% CO2. Cells tested negative for Mycoplasma contamination and were authenticated by a short tandem repeat profile using the AmpFISTR Identifier kit (Applied Biosystems).
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7

Neuroblastoma Cell Line Characterization

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All neuroblastoma cell lines were obtained from the Children’s Hospital of Philadelphia cell line bank, the Children’s Oncology Group, or ATCC. They were maintained in DMEM or RPMI-1640 supplemented with 10% FBS, 20 mM L-glutamine and antibiotics. 293FT cells were maintained in DMEM supplemented with 10% FBS and antibiotics. We received the cell lines in 2012 and the experiments were performed between 2012 and 2017. The genomic identity of each line was routinely tested and last confirmed 2015 using the AmpFISTR Identifier kit (Applied Biosystems). In addition, lines were routinely tested to confirm lack of mycoplasma contamination.
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8

Glioma Initiating Cells Isolation and Characterization

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Glioma initiating Cells (GICs) were established by isolating neurosphere-forming cells from surgical specimens of human GBM, as described previously [9 (link)]. These GICs were cultured as GBM neurospheres in DMEM/F12 medium containing B27 supplement (Invitrogen, Carlsbad, CA) and basic fibroblast growth factor and epidermal growth factor (20 ng/ml each). This study was approved by the institutional review board of The University of Texas MD Anderson Cancer Center (Houston, TX, USA). Both unsupervised and supervised approaches were used to classify GICs into molecular subtypes [5 (link)]. Cells were authenticated by testing short tandem repeats (STR) using the Applied Biosystems AmpFISTR Identifier kit (Foster City, CA). The last authentication testing was done in March 2014. Glioma cell lines were grown in DMEM supplemented with 10% FBS. The TGFβR1 kinase inhibitor (LY 2157299) was purchased from Selleck Chemicals (Houston, TX) and was dissolved in dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO, USA) to a concentration of 10 mmol/L. Recombinant human TGFβ1 was purchased from R&D Systems (Minneapolis, MN, USA), reconstituted in 20 μg/mL in sterile 4 mM HCl containing 1 mg/mL bovine serum albumin and stored at −20°C and further diluted to an appropriate final concentration in DMEM/F12 medium at the time of use.
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9

Multiple Myeloma Cell Lines Characterization

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The following human MM cell lines were obtained from ATCC (Rockville, MD, USA): RPMI8226, U266, and NCI-H929. The CAG MM cell line (generated by the group at the University of Arkansas for Medical Sciences (UAMS) [26 (link)]), OPM-1, and OPM-2 (originate from the same individual) were kindly provided by Prof. Israel Vlodavsky, Technion, Israel. Cells were maintained in log-phase growth in RPMI1640 medium (Biological Industries) supplemented with 10% heat-inactivated fetal calf serum (FCS), 1 mM L-glutamine, 100 U/ml penicillin, 0.01 mg/ml streptomycin, and 1 mM sodium pyruvate (Biological Industries) in a humidified atmosphere of 5% CO2 at 37 °C. U266 cells were authenticated in 2017 by short tandem repeat (STR) DNA profiling using AmpFISTR Identifier Kit (Applied Biosystems) and other cell lines were authenticated in 2021 at the Genomics Center of Biomedical Core Facility, Technion using the Promega GenePrint 24 System.
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10

Characterizing FLT3 Mutations in Leukemia Cell Lines

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The human AML cell lines MOLM14, MV4-11 (harboring FLT3-ITD mutations), OCI-AML3 and THP-1 (harboring FLT3 wildtype [WT]); the murine leukemia cell lines Ba/F3 (harboring different FLT3 mutations including ITD, TKD or ITD+TKD double mutations or FLT3 WT); and MSC were used for this study. Details of the cell lines and culture conditions are provided in the Online Supplementary Methods. All cell lines were validated by STR DNA fingerprinting using the AmpFISTR Identifier kit according to the manufacturer's instructions (Applied Biosystems cat. 4322288).
AML patients’ samples were obtained after written informed consent following institutional guidelines of the University of Texas MD Anderson Cancer Center and in accordance with the principles of the Declaration of Helsinki. Mononuclear cells were purified from primary samples by Ficoll-Hypaque (Sigma-Aldrich) density-gradient centrifugation and were cultured in RPMI 1640 culture medium supplemented with 10% fetal bovine serum before treatment.
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