Human non-transformed breast epithelial MCF10A cells, human breast cancer MCF7, SKBR3, T74D, MDA-MB-231 cells and murine mammary cancer JC cells were purchased from ATCC (Manassas, VA). Murine mammary cancer TUBO cells were a kind gift of Prof. Federica Cavallo, Department of Molecular Biotechnology and Health Sciences, University of Turin, Italy. Cells were maintained in medium supplemented with 10% v/v fetal bovine serum, 1% v/v penicillin-streptomycin, 1% v/v L-glutamine. 2 × 105 JC cells were stably transfected with 2 μg of pGL4.51[luc2/CMV/Neo] vector (Promega Corporation, Madison, WI), then selected in culture medium containing 1 μg/mL neomycin to generate the stably luciferase-expressing JC-luc clone.
Pgl4.51 luc2 cmv neo vector
Manufactured by Promega
Sourced in United States
The PGL4.51[luc2/CMV/Neo] vector is a plasmid that contains the luc2 luciferase gene under the control of the CMV promoter, along with a neomycin resistance gene. This vector is designed for use in eukaryotic cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (3 mentions)
L glutamine, by Thermo Fisher Scientific (3 mentions)
Mda mb 231, by American Type Culture Collection (2 mentions)
Gene pulser electroporator, by Bio-Rad (2 mentions)
Stemelite id system, by Promega (2 mentions)
Nivo s micro plate reader, by PerkinElmer (2 mentions)
Nano glo dual luciferase reporter assay system, by Promega (2 mentions)
Dbtrg, by Promega (2 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (2 mentions)
Dbtrg, by American Type Culture Collection (2 mentions)
Mcf 10a, by American Type Culture Collection (1 mentions)
Mcf 7, by American Type Culture Collection (1 mentions)
Skbr3, by American Type Culture Collection (1 mentions)
Pdonr221 vector, by Thermo Fisher Scientific (1 mentions)
293ft cell, by Thermo Fisher Scientific (1 mentions)
Bp clonase, by Thermo Fisher Scientific (1 mentions)
Lr clonase, by Thermo Fisher Scientific (1 mentions)
Insulin transferrin selenium, by Thermo Fisher Scientific (1 mentions)
Nu serum, by Corning (1 mentions)
G418 antibiotic, by Thermo Fisher Scientific (1 mentions)
22 protocols using pgl4.51 luc2 cmv neo vector
1
Breast Cancer Cell Culture
2
Lentiviral Vector Construction and Titration
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The previously described pSMAL vector modified from the MA1 lentiviral vector to have a Gateway cassette and SFFV promoter was used as the backbone for all generated lentiviral vectors49 (link),50 (link). HIST1H3H WT/K27M and HIST1H3F WT/K27I with a C-terminal HA-tag and attB sites for Gateway cloning were generated using GeneArt™ DNA Strings Fragments (Life Technologies). Luciferase modified for human expression (Luc2) was amplified out of the pGL4.51[luc2/CMV/NEO] vector (Promega). Genes were cloned into the pDONR221 vector (Invitrogen) using BP Clonase (Invitrogen) and subsequently cloned into the pSMAL vector using LR Clonase (Invitrogen) as per the manufacturer’s instructions. Lentiviral particles were produced in 293FT cells (Life Technologies) as previously. Viral titer was determined by adding serial dilutions of the concentrated viral particles onto 8227 AML cells and the percentage of GFP+ cells was measured 96 h later by flow cytometry.
3
Adrenocortical Carcinoma Cell Lines
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Two human ACC cell lines, SW13 and NCI-H295R, were purchased from the American Type Culture Collection™ (Cat # CCL-105, CRL-2128; Manassas, VA, USA) and cultured in 5% CO2 atmosphere at 37 °C in Dulbecco's Modified Eagle Medium (Cat # 11195–065, Thermo Fisher Scientific, MA, USA) supplemented with 2.5% Nu-Serum (Cat # 355100, Corning, MA, USA) and 0.1% Insulin-Transferrin-Selenium (Cat # 41400045, Thermo Fisher Scientific, MA, USA). Cell lines were authenticated by short tandem repeat profiling. We routinely subcultured every 3–5 days, depending on the degree of cell confluence.
NCI-H295R cells used to generate human ACC xenograft were transfected with a linearized pGL4.51[luc2/CMV/Neo] vector (9PIE132, Promega) encoding the luciferase reporter gene luc2 (Photinus pyralis) and maintained in the above medium with up to 500 μg/mL of G-418 antibiotic (11811–023, Gibco, MA, USA) for selection.
NCI-H295R cells used to generate human ACC xenograft were transfected with a linearized pGL4.51[luc2/CMV/Neo] vector (9PIE132, Promega) encoding the luciferase reporter gene luc2 (Photinus pyralis) and maintained in the above medium with up to 500 μg/mL of G-418 antibiotic (11811–023, Gibco, MA, USA) for selection.
4
Bioluminescent Imaging of Xenografts
5
Generating Luciferase-Expressing Prostate Cancer Cell Lines
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The PC3 prostate cancer cell line (Prostate Specific Antigen (PSA) non-expressing, androgen-independent) (ATCC, Manassas, VA, USA) was stably transfected with a firefly luciferase gene luc2 (pGL4.51 [luc2/CMV/Neo] vector, Promega, Madison, WI, USA) using a Gene PulserTM electroporator (Bio-Rad, CA, USA). LNCaP (PSA expressing, androgen-sensitive) were purchased from ATCC, and the LNCaP-derived C4-2B4 strain was supplied by the University of Bern (Bern, Switzerland). All cell lines were maintained in Dulbecco’s Modified Eagle’s Medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), supplemented with antibiotics and foetal bovine serum (FBS) (Sigma Aldrich, St. Louis, MO, USA). All cell lines were genetically profiled by Stem Elite ID system (Promega, Madison, WI, USA), which confirmed their identity (18/18 STRs) and regularly screened for mycoplasma contamination.
6
Establishment and Characterization of PDAC Cell Lines
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The human PDAC cell lines AsPC‐1 and BxPC‐3 were obtained from the American Type Culture Collection. PK‐45H, PK‐59, MIA Paca2, PANC‐1, and PK‐1 were obtained from RIKEN BRC through the National Bio‐Resource Project of MEXT. KP‐2, KP‐3L, KP‐4, and SUIT‐2 were obtained from the Japanese Collection of Research Bioresources. SNU‐213 and SNU‐324 were obtained from the Korean Cell Line Bank. TCC‐Pan2, PSN‐1, Sui65, Sui66, Sui67, Sui68, Sui69, Sui70, Sui71, Sui72, Sui73, Sui74, Sui75, and Sui76 were established by Yanagihara et al. at the National Cancer Center (Chiba, Japan).16 , 17 , 18 All cell lines were authenticated by the providers or by Yanagihara et al. for the short‐tandem repeat profile. After arrival, all cell lines were propagated and frozen immediately, and cells recovered from the frozen stock were used within 10 weeks. MIA Paca2 constitutively expressing the luciferase gene (referred to as MIA/luc) was generated by introducing the pGL4.51[luc2/CMV/Neo] vector (Promega) into MIA Paca2. PD0325901‐resistant MIA/luc cells were established by culturing the parent MIA/luc cells for 1 month with gradually increasing concentrations of PD0325901 (from 20 to 80 nM).
7
Isolation and Expansion of Pancreatic MSCs
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MSCs were isolated from healthy human pancreatic ductal tissue and expanded ex vivo as previously described (31 (link)). Briefly, human pancreases were obtained with full informed consent from adult heart-beating cadaver organ donors through the organ procurement program at the British Columbia Transplant Society (Vancouver, Canada). A total of 5 pancreatic ductal tissue samples were collected between July 2011 and November 2011 at Vancouver General Hospital (Vancouver, Canada). The pancreatic ductal tissue taken from the Ricordi® Chamber (Biorep Technologies, Inc., Miami, FL, USA), which was used for islet isolation, was utilized as the starting material for MSC isolation. The human glioma U251 cell line was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) to be used as target cells in the present study. U251 cells were maintained as suggested by ATCC, and the culture conditions were kept consistent with those of the MSCs. In order to track the viability in a timely manner, the U251 cells were pre-labeled with luciferase using the pGL4.51[luc2/CMV/Neo] vector (Promega Corporation, Madison, WI, USA), according to the manufacturer's protocol.
8
Glioblastoma Cell Lines and Primary Cultures
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Glioblastoma U87, C6, and Hs683 cells were obtained from American Type Culture Collection and GBM8401 was obtained from Bioresource Collection and Research Center of Taiwan. Those cell lines were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (Biological Industries). Primary cortical neuron and astrocyte cultures were prepared as previously described [44 (link)]. The human glioma cell lines were authenticated through cell morphology monitoring, growth curve analysis and short tandem repeat profiling analysis in 2014. The characterization of primary cortical neuron, primary astrocyte and C6 glioma cell line were confirmed by NeuN, GFAP or S100 expression, respectively. For the intracranial xenograft experiment, luciferase-expressing U87 and GBM8401 cells were established by transfection of a pGL4.51[luc2/CMV/Neo] vector (Promega) into the cell lines.
9
Characterization of B-ALL Cell Lines
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NALM-6 (B-ALL), RS4;11 (KMT2A-rearranged B-ALL) and SUP-B15 (Ph-like B-ALL) were purchased from ATCC. NALM-6 and RS4;11 were cultured in RPMI 1640 (Corning) supplemented with 10% fetal bovine serum (FBS) (GeminiBio), 2 mmol/L L-glutamine (Gibco), and 100 mg/mL streptomycin/100 U/mL penicillin (Lonza). SUP-B15 cells were cultured in McCoy 5A (modified) media (Gibco) supplemented with 20% FBS, 100 mg/mL streptomycin and 100 U/mL penicillin. RS4;11 cells were transduced to express the luminescent reporter luciferase using pGL4.51[luc2/CMV/Neo] vector (Promega). Cell authentication was performed using short tandem repeat analysis (Idexx BioAnalytics, Westbrook, ME) and per ATCC guidelines using morphology, growth curves, and Mycoplasma testing within 6 months of use (InVivoGen). Cell lines were maintained in culture at 37°C in 5% CO2.
10
Generating Luciferase-Expressing U-87MG Cells
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Luciferase-expressing U-87MG cells are established by transfection of a pGL4.51 [luc2/CMV/Neo] vector (Promega, Madison, WI, USA) into the human glioblastoma cell line U-87MG. The culture medium was changed to the complete medium supplemented with 10% FBS and 1% PS, 17 h after the transfection. Stable cells were selected in the culture medium. The resulting cell line is referred to Luc-U87MG 21 (link).
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