荧光素酶标记癌细胞的体内生物发光成像

Marc Carceles-Cordon; Irene Rodriguez-Fernandez; Veronica Rodriguez-Bravo; Carlos Cordon-Cardo; Josep Domingo-Domenech

doi:10.21769/BioProtoc.1762

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Peer-reviewed

In vivo Bioluminescence Imaging of Luciferase-labeled Cancer Cells

荧光素酶标记癌细胞的体内生物发光成像

Marc Carceles-Cordon

Irene Rodriguez-Fernandez

Veronica Rodriguez-Bravo

CC Carlos Cordon-Cardo

Josep Domingo-Domenech email

发布: 2016年03月20日第6卷第6期 DOI: 10.21769/BioProtoc.1762 浏览次数: 35062

评审: Vivien Jane Coulson-ThomasAnonymous reviewer(s)

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Cover of Cancer Cell, featuring study using the protocol.

Feb 2015

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Abstract

Over the past decade, in vivo bioluminescent imaging has emerged as a non-invasive and sensitive tool for studying ongoing biological processes within living organisms (Contag et al., 1997; Contag et al., 1998). Based on the detection and quantitation of the photons produced by the oxidation of luciferin by luciferase enzymes (Harvey, 1927), this technique has proved to be particularly useful in analyzing cancerous cells and monitoring tumor growth (Edinger et al., 1999; Sweeney et al., 1999; Vidal et al., 2015), providing a cost-effective insight into how the disease progresses in vivo, without the need of serial sacrifice of animals. This protocol describes in detail the procedure of obtaining luciferase-tagged tumors in immunocompromised mice that can be studied by bioluminescent imaging through the use of an IVIS Spectrum imager.

Keywords: Bioluminescent imaging (生物发光成像)

Materials and Reagents

Falcon^TM Standard 10 cm² Tissue Culture Dishes (Corning, catalog number: 353003 )
Syringe Filter 0.45 μm strainer (Corning, catalog number: CLS431225 )
22Rv1 prostate cancer cells (ATCC, catalog number: CRL-2505 )
NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NSG^TM) mice (Jackson Lab, catalog number: 00557 )
Phoenix-Ampho cells (ATCC, catalog number: CRL-3213 )
pLenti CMV Puro LUC (w168-1) (Addgene, catalog number: 17477 )
Helper pCMV-VSV-G (Addgene, catalog number: 8454 ) and pMD2.G (Addgene, catalog number: 12259 )
One Shot Stbl3 Competent E. coli (Thermo Fisher Scientific, Invitrogen^TM, catalog number: C7373-03 )
LB Broth Medium (Thermo Fisher Scientific, BioReagents^TM, catalog number: BP1426-2 )
Ampicillin (Sigma-Aldrich, catalog number: A9393 )
QIAprep Spin Miniprep Kit (QIAGEN, catalog number: 27106 )
RPMI 1640 Medium (Thermo Fisher Scientific, Gibco^TM, catalog number: 11875119 )
Dulbecco’s Phosphate-Buffered Saline (DPBS) 1x (Corning, catalog number: 21-031 )
jetPEI^® DNA Transfection Agent (Polyplus-transfection, catalog number: 101-10N )
Penicillin-Streptomycin antibiotic (Thermo Fisher Scientific, Gibco^TM, catalog number: 15140122 )
Polybrene^® (Santa Cruz Biotechnology, catalog number: 134220 )
Isoflurane (Baxter, catalog number: 1001936040 )
Matrigel^® Growth Factor Reduced (GFR) Basement Membrane Matrix (Corning, catalog number: 354230 )
D-luciferin (PerkinElmer, catalog number: 770504 )

Equipment

Thermo-block (Thermo Fisher Scientific, model: Isotemp^TM Digital and Analog Dry Bath Incubator )
Shaker incubator (Eppendorf AG, New Brunswick Scientific, model: Innova 44R Incubator/Shaker )
Table-top microcentrifuge (Eppendorf AG, model: centrifuge 5415R )
Spectrophotometer (Thermo Fisher Scientific, model: SPECTRONIC^TM 200 Spectrophotometer )
Incubator (Thermo Fisher Scientific, model: Heracell 150i )
Vortex (Scientific Industries, model: Vortex Genie 2 )
IVIS Spectrum imager (PerkinElmer, model: IVIS Spectrum Preclinical In Vivo Imaging System )

Software

Caliper LifeScience Living Image^® in vivo imaging software (PerkinElmer, catalog number: 128110) (Lumina/Kinetic/XR/100, Living Image V4.1)

Figure 1. Schematic representation of the steps required to successfully tag cells with luciferase and perform in vivo imaging

Procedure

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如何引用

Carceles-Cordon, M., Rodriguez-Fernandez, I., Rodriguez-Bravo, V., Cordon-Cardo, C. and Domingo-Domenech, J. (2016). In vivo Bioluminescence Imaging of Luciferase-labeled Cancer Cells. Bio-protocol 6(6): e1762. DOI: 10.21769/BioProtoc.1762.