搜索

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   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

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), In vivo(在体内), Mice(老鼠), Cancer cells(癌细胞), Tumour(肿瘤)

Materials and Reagents

  1. FalconTM Standard 10 cm2 Tissue Culture Dishes (Corning, catalog number: 353003 )
  2. Syringe Filter 0.45 μm strainer (Corning, catalog number: CLS431225 )
  3. 22Rv1 prostate cancer cells (ATCC, catalog number: CRL-2505 )
  4. NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSGTM) mice (Jackson Lab, catalog number: 00557 )
  5. Phoenix-Ampho cells (ATCC, catalog number: CRL-3213 )
  6. pLenti CMV Puro LUC (w168-1) (Addgene, catalog number: 17477 )
  7. Helper pCMV-VSV-G (Addgene, catalog number: 8454 ) and pMD2.G (Addgene, catalog number: 12259 )
  8. One Shot Stbl3 Competent E. coli (Thermo Fisher Scientific, InvitrogenTM, catalog number: C7373-03 )
  9. LB Broth Medium (Thermo Fisher Scientific, BioReagentsTM, catalog number: BP1426-2 )
  10. Ampicillin (Sigma-Aldrich, catalog number: A9393 )
  11. QIAprep Spin Miniprep Kit (QIAGEN, catalog number: 27106 )
  12. RPMI 1640 Medium (Thermo Fisher Scientific, GibcoTM, catalog number: 11875119 )
  13. Dulbecco’s Phosphate-Buffered Saline (DPBS) 1x (Corning, catalog number: 21-031 )
  14. jetPEI® DNA Transfection Agent (Polyplus-transfection, catalog number: 101-10N )
  15. Penicillin-Streptomycin antibiotic (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  16. Polybrene® (Santa Cruz Biotechnology, catalog number: 134220 )
  17. Isoflurane (Baxter, catalog number: 1001936040 )
  18. Matrigel® Growth Factor Reduced (GFR) Basement Membrane Matrix (Corning, catalog number: 354230 )
  19. D-luciferin (PerkinElmer, catalog number: 770504 )

Equipment

  1. Thermo-block (Thermo Fisher Scientific, model: IsotempTM Digital and Analog Dry Bath Incubator )
  2. Shaker incubator (Eppendorf AG, New Brunswick Scientific, model: Innova 44R Incubator/Shaker )
  3. Table-top microcentrifuge (Eppendorf AG, model: centrifuge 5415R )
  4. Spectrophotometer (Thermo Fisher Scientific, model: SPECTRONICTM 200 Spectrophotometer )
  5. Incubator (Thermo Fisher Scientific, model: Heracell 150i )
  6. Vortex (Scientific Industries, model: Vortex Genie 2 )
  7. IVIS Spectrum imager (PerkinElmer, model: IVIS Spectrum Preclinical In Vivo Imaging System )

Software

  1. 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

  1. Bacterial transformation
    Artificially constructed plasmids are the most commonly used vectors for introducing foreign DNA into bacterial cells. In this protocol, we used the commercial plasmid Lenti CMV Puro LUC, which contains the firefly luciferase gene, and was transformed into a culture of Stbl3 competent cells.
    1. Transfer 50 μl of Stbl3 competent cells in a 1.5 ml Eppendorf tube placed on ice.
    2. Add between 100 ng and 500 ng of plasmid into the tube and resuspend with Stbl3 competent cells (pipet to resuspend, do not use vortex).
    3. Incubate the tube on ice for 30 min, gently resuspend every 10 min.
    4. Place the tube in a thermo-block for exactly 45 sec at 42 °C, and immediately put back into ice for 2 min.
    5. Add 800 μl of LB-Medium with no antibiotic into the tube, and gently resuspend the sample with a pipet (do not use vortex).
    6. Grow up bacteria in a shaker incubator at 37 °C speed 200 rpm for 2-3 h.
    7. Plate 100 µl of transformation mixture on LB-agar plates with 100 μg/ml of ampicillin.
    8. Place plate in an incubator at 37 °C overnight.
    9. Pick a single colony and inoculate a flask containing 50 ml of LB-Medium + 100 μg/ml of ampicillin.
    10. Incubate overnight at 37 °C in a shaker incubator at a speed of 200 rpm.

  2. DNA extraction
    There are several methods that can be used to purify plasmid DNA depending on the size of the bacterial culture and its corresponding plasmid yield, and a handful of kits available from varying manufacturers to do it. Here, we perform a minipreparation (miniprep) using the QIAprep Spin Miniprep Kit, which allows the isolation of a small-scale sample of up to 20 μg plasmid DNA from the transformed bacteria. Briefly, in this protocol the isolation of DNA from bacteria relies upon the use of columns and centrifugation steps in which DNA is sheared, extracted, and precipitated. Follow protocol of the manufacturer.

  3. Transfection of DNA into viral packaging cells
    Depending on the plasmid, several packaging cells can be used to generate the desired virus. In this protocol we used Phoenix-Ampho cells, which are a second-generation retrovirus producer cell line that is also capable of a long-term stable production of lentiviruses when helper DNA (CMV and DMV) is present. Helper DNA encodes viral genes that increase viral replication and packaging.
    1. Seed 3 x 106 Phoenix-Ampho cells in 8 ml of RPMI supplemented with 10% FBS and 1% PenStrep in a 10 cm2 culture plate 24 h before transfection.
    2. In a first 1.5 ml Eppendorf tube, add 6 μg of DNA, 3 μg of each helper (CMV and DMV) and fill up to 250 μl of 150 mM NaCl (per sample to transfect).
    3. In a second 1.5 ml tube add 24 μl of jetPEI and 226 μl of 150 mM NaCl (per sample to transfect).
    4. Vortex the tubes for 10 sec.
    5. Add the Jetpei + NaCl into the Eppendorf tube with the DNA and vortex for 10 sec.
    6. Incubate mixture for 15 min at room temperature.
    7. Remove media of plate and add 9.5 ml of fresh RPMI supplemented with 10% FBS and 1% PenStrep to Phoenix-Ampho cells (see step C1).
    8. Add 500 μl drop by drop, of the mixture (step C5) to the plate and incubate overnight at 37 °C.
    9. After 24 h change the medium and add fresh RPMI supplemented with 10% FBS and 1% PenStrep.
    10. After 48 h, collect and filter the medium with a 0.45 μm strainer into a 50 ml Falcon tube.
      Note: Viral concentration can be measured after this step by serial dilution to obtain quantitative information. Various techniques can be used to quantify virus concentration which include traditional methods (e.g. plaque assay) and commercial kits (e.g., ELISA or Q-PCR).

  4. Viral particle cell infection
    The viral particles produced in the previous step are used to infect cells. Virus will infect cells which hold a receptor that the virus can bind to. Therefore, the efficiency of viral infection will greatly depend on the cell type. In this protocol, we infected 70% confluent 22Rv1 prostate cancer cells growing in 10 cm2 culture dishes with 10 ml of media.
    1. Add Polybrene to cultured cells to a final concentration of 10 μg/ml.
      Note: Polybrene is an efficient infection reagent used to introduce viral vectors into mammalian cells. Polybrene acts by neutralizing the charge repulsion between virions and the cell surface (Davis et al., 2004).
    2. Using a 5 ml pipet, add 4 ml of filtered viral particles to cultured cells.
    3. Centrifuge 10 cm2 plate at 250 x g for 5 min.
      Note: This step helps the physical contact of viruses with surface of cells increasing infection efficiency.
    4. Place cells in 37 °C, 5% CO2 incubator for 18-24 h.
      Note: Infection efficiency can be affected by incubation time. Troubleshooting using different incubation times can be performed to identify most efficient incubation time for a specific cell type.
    5. Aspirate the medium and add 10 ml of fresh RPMI supplemented with 10% FBS and 1% PenStrep. Incubate at 37 °C for 24 h.
    6. Select infected 22Rv1 prostate cancer cells by adding Puromycin at a final concentration of 2 µg/ml to culture media.
      Note: Puromycin concentration will depend on cell type.
    7. Replace medium containing Puromycin every 3 days. Cells that have incorporated the pLenti CMV Puro LUC will grow up under antibiotic selection conditions.
    8. Check that the cells have incorporated the luciferase plasmid.
      Note: Multiple methods can be used to test successful luciferase. Examples are detection of luciferase by PCR or measuring luciferase activity upon D-Luciferin exposure (see Figure 2).

  5. Injection of luciferase-tagged cells into immunocompromised mice
    In this protocol, we performed subcutaneous injection of luciferase-tagged tumor cells. However, alternative injection sites such as the mouse prostate (orthotropic), which promotes a prostate microenvironment, and subrenal capsule injection, which enhances successful engraftment and preserves tumor heterogeneity, can be performed based on investigator preference (Hidalgo et al., 2014; Siolas et al., 2013).
    Note: Conduct all animal procedures in compliance with protocols approved by the institutional animal care committee. This protocol has been conducted at our institution under a specific Animal Care Committee in accordance and compliance with all relevant regulatory and institutional agencies, regulations and guidelines.
    1. Harvest attached cells by removing culture media, washing cells with 10 ml 1x DBPS two times and trypsinizing cells using 2 ml 0.05% Trypsin. Recover 2 ml trypsinized cells to 15 ml tube containing 4 ml RPM1 supplemented with 10% FBS and 1% PenStep.
    2. Count cells using a hemocytometer.
    3. Mix 106 cells suspended in 100 μl of RPMI supplemented with 100 μl of extracellular matrix (1:1 ratio) and place on ice.
    4. Anesthetize mice prior to subcutaneous injection in a chamber supplying 5% (v/v) inhaled isoflurane in 1 L/min of oxygen.
    5. Using a 25 gauge needle and a 1 ml syringe, inject 250 μl of cell and extracellular matrix suspension subcutaneously into the flanks of immunodeficient NSG mice.

  6. Bioluminescence imaging
    The IVIS Spectrum imager expresses the bioluminescent signal in photons per second and displays it as an intensity map. The luminescence, which is the consequence of the photon flux emitted by the luciferase-expressing cells, directly correlates to the size of the tumor and can be measured at the site of injection using a region of interest (ROI) tool. The optical emission image can be adjusted to provide optimal contrast and resolution without affecting quantitation using the Living Image© in vivo imaging software.
    1. Prepare a fresh stock solution of D-Luciferin at 15 mg/ml in 1x DPBS.
    2. Anesthetize the mice using isoflurane vaporizer and place them inside the camera box of the IVIS Spectrum imager.
    3. Run a background image of the mice.
    4. Place the mice in dorsal recumbency (abdomen face up) and inject 150 mg/kg of D-Luciferin (from step F1) intra-peritoneally (ip) 5 min before imaging. The preferred region of injection is the mouse lower right quadrant of the abdominal region.
    5. Taking into account the time dependent D-Luciferin uptake by tumor cells, run sequential images of the mice every 2 min until luminescence saturation is reached.


      Figure 2. Image illustrates the IVIS spectrum imager and a mouse placed inside the camera box

Representative data


Figure 3. Luciferase detection in cells. Multiple methods can be used for testing the success of cell infection, which include but are not limited to: A. Genetic detection of luciferase gene by PCR and/or B. Testing luciferase activity.


Figure 4. Subcutaneous injection of cells in mice. A. Lift the skin over the back of the neck to make a tent. B. Insert the needle at the tent base, holding it parallel to the animal’s body to avoid puncturing underlying structures. C. Aspirate to create a light vacuum and ensure that the needle has not entered a blood vessel. Slowly inject the cell and extracellular matrix suspension.


Figure 5. Bioluminescence in vivo imaging. A. Representative luciferase time course (every 2 min) imaging of mice bearing subcutaneous tumors injected with luciferin. B. Graph illustrates the bioluminescence saturation of the whole mouse (displayed in A.) obtained until time point 8 min, when the experiment was decided to be concluded.

Acknowledgments

We thank the TJ Martell Foundation and Agilent Technologies for their support.

References

  1. Contag, C. H., Spilman, S. D., Contag, P. R., Oshiro, M., Eames, B., Dennery, P., Stevenson, D. K. and Benaron, D. A. (1997). Visualizing gene expression in living mammals using a bioluminescent reporter. Photochem Photobiol 66(4): 523-531.
  2. Contag, P. R., Olomu, I. N., Stevenson, D. K. and Contag, C. H. (1998). Bioluminescent indicators in living mammals. Nat Med 4(2): 245-47.
  3. Davis, H. E., Rosinski, M., Morgan, J. R. and Yarmush, M. L. (2004). Charged polymers modulate retrovirus transduction via membrane charge neutralization and virus aggregation. Biophys J 86(2): 1234-1242.
  4. Edinger, M., Sweeney, T. J., Tucker, A. A., Olomu, A. B., Negrin, R. S. and Contag, C. H. (1999). Noninvasive assessment of tumor cell proliferation in animal models. Neoplasia 1(4): 303-10.
  5. Harvey, E. N. (1927). The oxidation-reduction potential of the luciferin-oxyluciferin system. J Gen Physiol 10(3): 385-393.
  6. Hidalgo, M., Amant, F., Biankin, A. V., Budinska, E., Byrne, A. T., Caldas, C., Clarke, R. B., de Jong, S., Jonkers, J., Maelandsmo, G. M., Roman-Roman, S., Seoane, J., Trusolino, L. and Villanueva, A. (2014). Patient-derived xenograft models: an emerging platform for translational cancer research. Cancer Discov 4(9): 998-1013.
  7. Siolas, D. and Hannon, G. J. (2013). Patient-derived tumor xenografts: transforming clinical samples into mouse models. Cancer Res 73(17): 5315-19.
  8. Sweeney, T. J., Mailander, V., Tucker, A. A., Olomu, A. B., Zhang, W., Cao, Y., Negrin, R. S. and Contag, C. H. (1999). Visualizing the kinetics of tumor-cell clearance in living animals. Proc Natl Acad Sci U S A 96(21): 12044-12049.
  9. Vidal, S. J., Rodriguez-Bravo, V., Quinn, S. A., Rodriguez-Barrueco, R., Lujambio, A., Williams, E., Sun, X., de la Iglesia-Vicente, J., Lee, A., Readhead, B., Chen, X., Galsky, M., Esteve, B., Petrylak, D. P., Dudley, J. T., Rabadan, R., Silva, J. M., Hoshida, Y., Lowe, S. W., Cordon-Cardo, C. and Domingo-Domenech, J. (2015). A targetable GATA2-IGF2 axis confers aggressiveness in lethal prostate cancer. Cancer Cell 27(2): 223-239.

简介

在过去十年中,体内生物发光成像已经成为用于研究活生物体内正在进行的生物过程的非侵入性和敏感性工具(Contag等人,1997; Contag et al。,1998)。 基于通过荧光素酶氧化荧光素产生的光子的检测和定量(Harvey,1927),该技术已经证明在分析癌细胞和监测肿瘤生长中特别有用(Edinger等, ,1999; Sweeney等人,1999; Vidal等人,2015),提供了关于疾病在体内如何进展的成本效益的洞察 ,无需连续牺牲动物。 该协议详细描述了在免疫受损的小鼠中获得荧光素酶标记的肿瘤的程序,其可以通过使用IVIS光谱成像仪通过生物发光成像进行研究。

关键字:生物发光成像, 在体内, 老鼠, 癌细胞, 肿瘤

材料和试剂

  1. 组织培养皿(Corning,目录号:353003)的标准10cm TM
  2. 注射器过滤器0.45μm过滤器(Corning,目录号:CLS431225)
  3. 22Rv1前列腺癌细胞(ATCC,目录号:CRL-2505)
  4. NOD.Cg- /SzJ(NSG )小鼠(Jackson Lab,目录号:00557)
  5. Phoenix-Ampho cell(ATCC,目录号:CRL-3213)
  6. pLenti CMV Puro LUC(w168-1)(Addgene,目录号:17477)
  7. 辅助pCMV-VSV-G(Addgene,目录号:8454)和pMD2.G(Addgene,目录号:12259)
  8. One Shot Stbl3胜任力大肠杆菌(Thermo Fisher Scientific,Invitrogen TM ,目录号:C7373-03)
  9. LB肉汤培养基(Thermo Fisher Scientific,BioReagents TM ,目录号:BP1426-2)
  10. 氨苄青霉素(Sigma-Aldrich,目录号:A9393)
  11. QIAprep Spin Miniprep Kit(QIAGEN,目录号:27106)
  12. RPMI 1640 Medium(Thermo Fisher Scientific,Gibco TM ,目录号:11875119)
  13. Dulbecco磷酸盐缓冲盐水(DPBS)1x(Corning,目录号:21-031)
  14. jetPEI DNA转染剂(Polyplus转染,目录号:101-10N)
  15. 青霉素 - 链霉素抗生素(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  16. Polybrene (Santa Cruz Biotechnology,目录号:134220)
  17. 异氟烷(Baxter,目录号:1001936040)
  18. 生长因子减少(GFR)基底膜基质(Corning,目录号:354230)
  19. D-荧光素(PerkinElmer,目录号:770504)

设备

  1. Thermo-block(Thermo Fisher Scientific,型号:Isotemp 数字和模拟干浴培养箱)
  2. 摇床孵育器(Eppendorf AG,New Brunswick Scientific,型号:Innova 44R孵育器/振荡器)
  3. 台式微量离心机(Eppendorf AG,型号:离心机5415R)
  4. 分光光度计(Thermo Fisher Scientific,型号:SPECTRONIC 200分光光度计)
  5. 孵育器(Thermo Fisher Scientific,型号:Heracell 150i)
  6. Vortex(Scientific Industries,型号:Vortex Genie 2)
  7. IVIS光谱成像仪(PerkinElmer,型号:IVIS Spectrum临床前体内成像系统)

软件

  1. Caliper LifeScience Living Image 成像软件(PerkinElmer,目录号:128110)(Lumina/Kinetic/XR/100,Living Image V4.1)


    图1.使用萤光素酶成功标记细胞并在体内执行 成像所需的步骤的示意图。

程序

  1. 细菌转化
    人工构建的质粒是用于将外来DNA引入细菌细胞的最常用的载体。在该方案中,我们使用商业质粒Lenti CMV Puro LUC,其含有萤火虫荧光素酶基因,并转化到Stb13感受态细胞的培养物中。
    1. 转移50微升Stbl3感受态细胞在放置在冰上的1.5毫升的Eppendorf管
    2. 将100ng和500ng质粒加入试管中并重悬 与Stbl3感受态细胞(移液器重悬,不使用涡旋)。
    3. 孵育管在冰上30分钟,轻轻重悬每10分钟。
    4. 将管在热块中在42℃下精确45秒,并立即放回冰中2分钟。
    5. 加入800μl没有抗生素的LB培养基管,轻轻地用移液管重悬样品(不要使用涡流)。
    6. 在摇床培养箱中以37℃速度200rpm培养细菌2-3小时
    7. 将100μl转化混合物在含有100μg/ml氨苄青霉素的LB-琼脂平板上平板
    8. 将板在孵育器中37℃过夜
    9. 挑取单个菌落并接种到含有50ml LB-培养基+100μg/ml氨苄青霉素的烧瓶中。
    10. 在37℃下在振荡器孵育器速度200rpm中孵育过夜。

  2. DNA提取
    有几种方法可用于纯化质粒DNA,这取决于细菌培养物的大小及其相应的质粒产量,以及可从不同制造商获得的几种试剂盒。在这里,我们使用QIAprep Spin Miniprep Kit进行小量制备(miniprep),其允许从转化的细菌中分离最多20μg质粒DNA的小规模样品。简言之,在该方案中,从细菌中分离DNA依赖于柱的使用和离心步骤,其中DNA被剪切,提取和沉淀。按照制造商的协议。

  3. DNA转染到病毒包装细胞中 根据质粒,可以使用几种包装细胞来产生所需的病毒。在这个协议中,我们使用Phoenix-Ampho细胞,这是第二代逆转录病毒生产细胞系,当辅助DNA(CMV和DMV)存在时,也能够长期稳定生产慢病毒。辅助DNA编码增加病毒复制和包装的病毒基因。
    1. 种子3×10 6个Phoenix-Ampho细胞在8ml补充有10% FBS和1%PenStrep在10cm 2培养板中转染前24小时。
    2. 在第一个1.5ml Eppendorf管中,加入6μgDNA,每种3μg 辅助(CMV和DMV),并填充至250μl的150mM NaCl(每个样品至 ?转染)。
    3. 在第二个1.5ml管中加入24μl的jetPEI和226μl的150mM NaCl(每个样品进行转染)。
    4. 涡旋管10秒。
    5. 加入Jetpei + NaCl的Eppendorf管与DNA和涡流10秒。
    6. 在室温下孵育混合物15分钟。
    7. 取出板的培养基,加入9.5ml新鲜RPMI补充 ?10%FBS和1%PenStrep到Phoenix-Ampho细胞(参见步骤C1)
    8. 逐滴加入500μl混合物(步骤C5)至平板,并在37℃下孵育过夜。
    9. 24小时后更换培养基并加入补充有10%FBS和1%PenStrep的新鲜RPMI
    10. 48小时后收集并用0.45μm过滤器过滤介质 到50ml Falcon管中。
      注意:可以测量病毒浓度 之后通过连续稀释获得定量信息。 可以使用各种技术来量化病毒浓度 包括传统方法(例如 空斑测定)和商业试剂盒 ( ELISA或Q-PCR)。

  4. 病毒颗粒细胞感染
    在前面步骤中产生的病毒颗粒用于感染细胞。病毒将感染具有病毒可结合的受体的细胞。因此,病毒感染的效率将极大地取决于细胞类型。在该方案中,我们用10ml培养基感染生长在10cm 2培养皿中的70%汇合的22Rv1前列腺癌细胞。
    1. 向培养的细胞中加入聚凝胺,使终浓度为10μg/ml。
      注意:Polybrene是一种有效的感染试剂,用于引入 病毒载体进入哺乳动物细胞。聚苯乙烯通过中和作用 病毒体和细胞表面之间的电荷排斥(Davis等人, 2004)。
    2. 使用5ml移液管,向培养的细胞中加入4ml过滤的病毒颗粒
    3. 在250×g离心10分钟2小时5分钟。
      注意:此步骤有助于病毒与细胞表面的物理接触,提高感染效率。
    4. 将细胞置于37℃,5%CO 2培养箱中18-24小时。
      注意:感染效率可能受孵育时间的影响。 可以使用不同的孵育时间进行故障排除 确定特定细胞类型的最有效的孵育时间。
    5. 吸出培养基并加入10ml补充有10%FBS和1%PenStrep的新鲜RPMI。在37℃孵育24小时
    6. 通过向培养基中加入终浓度为2μg/ml的嘌呤霉素选择感染的22Rv1前列腺癌细胞。
      注意:嘌呤霉素浓度取决于细胞类型。
    7. 每3天更换含有嘌呤霉素的培养基。细胞有 纳入pLenti CMV Puro LUC将在抗生素下生长 选择条件。
    8. 检查细胞是否掺入了荧光素酶质粒。
      注意:多种方法可用于测试成功的荧光素酶。 实例是通过PCR检测荧光素酶或测量荧光素酶 对D-荧光素暴露的活性(参见图2)。

  5. 将荧光素酶标记的细胞注射到免疫受损的小鼠中
    在该协议中,我们进行荧光素酶标记的肿瘤细胞的皮下注射。然而,可以基于研究者的偏好来进行替代的注射位点,例如促进前列腺微环境的小鼠前列腺(正交位点)和能够增强成功植入和保持肿瘤异质性的肾下膜注射(Hidalgo et al。/em>,2014; Siolas et al。,2013)。
    注意:按照机构动物护理委员会批准的方案进行所有动物程序。该协议已在我们的机构根据特定的动物护理委员会进行,并遵守所有相关的监管和机构机构,法规和准则。
    1. 通过去除培养基,收获附着的细胞,用10洗涤细胞 ml 1×DBPS两次,并使用2ml 0.05%胰蛋白酶胰蛋白酶化细胞。 恢复2毫升胰蛋白酶化细胞到15毫升管含有4毫升RPM1 补充有10%FBS和1%PenStep
    2. 使用血细胞计数器计数细胞。
    3. 混合悬浮于100μl补充有100μl细胞外基质(1:1比例)的RPMI中的10 6细胞并置于冰上。
    4. 在皮下注射之前麻醉小鼠,在1L /分钟的氧气中提供5%(v/v)吸入异氟烷的室中。
    5. 使用25号针和1毫升注射器,注射250微升的细胞 和细胞外基质悬浮液皮下注射到侧腹 免疫缺陷NSG小鼠。

  6. 生物发光成像
    IVIS光谱成像仪以每秒光子数表示生物发光信号,并将其显示为强度图。发光,其是荧光素酶表达细胞发射的光子通量的结果,直接与肿瘤的大小相关,并且可以使用感兴趣区域(ROI)工具在注射部位测量。可以调整光发射图像以提供最佳对比度和分辨率,而不影响使用Living Image< i/sup>体内成像软件的定量。
    1. 在1x DPBS中制备15mg/ml的D-荧光素的新鲜储备溶液。
    2. 使用异氟醚蒸发器麻醉小鼠,并将它们放在IVIS Spectrum成像仪的相机箱内。
    3. 运行鼠标的背景图像。
    4. 放置小鼠背部躺着(腹部朝上),并注射150 ?mg/kg D-荧光素(来自步骤F1)腹膜内(ip)5分钟 成像前。优选的注射区域是小鼠 右腹象限。
    5. 考虑到 时间依赖性D-荧光素被肿瘤细胞摄取,运行顺序图像 的小鼠每2分钟,直到达到发光饱和

      图2.图像显示了IVIS光谱成像仪和放置在相机盒内的鼠标

代表数据


图3.细胞中的荧光素酶检测多种方法可用于测试细胞感染的成功,包括但不限于:A.通过PCR和/或B的荧光素酶基因的遗传检测。测试荧光素酶活性

图4.小鼠皮下注射细胞。A.将皮肤从颈背上抬起,形成一个帐篷。 B.将针头插入帐篷底座,将其平行于动物的身体,以避免刺破下面的结构。 C.吸气以产生轻真空并确保针没有进入血管。缓慢注射细胞和细胞外基质悬浮液。


图5.生物发光体内成像。 A。具有注射荧光素的皮下肿瘤的小鼠的代表性荧光素酶时程(每2分钟)成像。 B.图示说明直到时间点8分钟,当实验决定结束时获得的整个小鼠(显示在A.中)的生物发光饱和度。

致谢

我们感谢TJ Martell基金会和安捷伦科技的支持。

参考文献

  1. Contag,C.H.,Spilman,S.D.,Contag,P.R.,Oshiro,M.,Eames,B.,Dennery,P.,Stevenson,D.K。和Benaron,D.A。(1997)。 使用生物发光记者可视化活哺乳动物中的基因表达。 Photochem Photobiol 66(4):523-531。
  2. Contag,P.R.,Olomu,I.N.,Stevenson,D.K.and Contag,C.H。(1998)。 生物哺乳动物中的生物发光指示器。 Nat Med 4 2):245-47。
  3. Davis,H.E.,Rosinski,M.,Morgan,J.R。和Yarmush,M.L。(2004)。 带电荷的聚合物通过膜电荷中和和病毒聚集调节逆转录病毒转导。生物物J 86(2):1234-1242。
  4. Edinger,M.,Sweeney,T.J.,Tucker,A.A.,Olomu,A.B.,Negrin,R.S.and Contag,C.H。(1999)。 在动物模型中无创性评估肿瘤细胞增殖 肿瘤 1(4):303-10。
  5. Harvey,E.N。(1927)。 萤光素 - 氧化荧光素系统的氧化还原电位。 Physiol 10(3):385-393。
  6. Hidalgo,M.,Amant,F.,Biankin,AV,Budinska,E.,Byrne,AT,Caldas,C.,Clarke,RB,de Jong,S.,Jonkers,J.,Maelandsmo,GM,Roman-Roman ,S.,Seoane,J.,Trusolino,L.and Villanueva,A。(2014)。 患者衍生的异种移植模型:翻译癌症研究的新兴平台 Cancer Discov 4(9):998-1013。
  7. Siolas,D。和Hannon,G.J。(2013)。 可视化活体动物中肿瘤细胞清除的动力学。 Proc Natl Acad Sci USA 96(21):12044-12049。
  8. Vidal,SJ,Rodriguez-Bravo,V.,Quinn,SA,Rodriguez-Barrueco,R.,Lujambio,A.,Williams,E.,Sun,X.,de la Iglesia-Vicente,J.,Lee, ,Readhead,B.,Chen,X.,Galsky,M.,Esteve,B.,Petrylak,DP,Dudley,JT,Rabadan,R.,Silva,JM,Hoshida,Y.,Lowe,SW,Cordon-Cardo ,C.and Domingo-Domenech,J。(2015)。 可靶向的GATA2-IGF2轴赋予致死性前列腺癌的侵袭性。癌症Cell 27(2):223-239。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
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.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。