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In vivo Lineage-tracing Studies in a Cancer Stem Cell Population in Neuroblastoma
体内谱系追踪研究成神经细胞瘤中的癌症干细胞群   

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Abstract

Tumors are comprised of heterogeneous subpopulations that may exhibit differing capacity for differentiation, self-renewal, and tumorigenicity. In vivo lineage-tracing studies are a powerful tool for defining the role of tumor subpopulations in tumor growth and as targets for therapeutic agents. This protocol describes using a neuroblastoma cancer cell line transduced with two different fluorescent proteins (GFP and tdTomato) to track the specific contributions of cells expressing the GCSF receptor (CD114+) or not (CD114-) on tumor growth in vivo.

Materials and Reagents

  1. Human neuroblastoma cell lines (NGP, NB-1691, IMR-32)
    Note: A cell line is transduced with two different fluorescent proteins, for example GFP (Clontech, catalog number: 632370 ) and tdTomato (Clontech, catalog number: 632534 ), such that there is a GFP positive line and a tdTomato line of the same cell type. In this manner, subpopulations of the same cell type (i.e. GCSF-R positive and GCSF-R negative cells) can be traced. If in vivo monitoring of tumor growth via bioluminescent imaging is desired, cell lines should also be transfected or virally transduced with commercially available vectors, e.g. pGL2-Control Vector (Promega Corporation) to express a luminescent reporter gene. For a detailed description and protocol of in vivo bioluminescent imaging, please refer to Reference 2.
  2. 4-6 week old female non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice (Taconic, model number: NODSCF , http://www.taconic.com/NODSC)
  3. 293T cells
  4. RPMI medium 1640 (Life Technologies, catalog number: 11875-101 )
  5. 10% (v/v) fetal bovine serum (FBS) (Life Technologies, catalog number: 16000044 )
  6. 1% (v/v) penicillin/streptomycin (Life Technologies, catalog number: 15140122 ) (100x P/S; final concentration 100 units/ml penicillin and 100 mg/ml streptomycin)
  7. 1% (v/v) of 100x L-glutamine (Life Technologies, catalog number: 25030081 ) (final concentration 2 mM)
  8. Phosphate-buffered saline (PBS) (sterile) (Life Technologies, catalog number: 70011069 )
  9. 0.25% trypsin/EDTA (Life Technologies, catalog number: 25200056 )
  10. Collagenase I (Sigma-Aldrich, catalog number: C0130 ) [prepare a solution containing 10,000 Collagenase Digestion Unites (CDU/ml) in PBS]
  11. Dispase II (Roche Diagnostics, catalog number: 04942078001 ) (prepare a solution containing 32 mg/ml Dispase II in PBS)
  12. DNase I (EMD Millipore, Calbiochem®, catalog number: 260913 ) (prepare a solution containing 5 MU/ml DNase I)
  13. FuGENE 6 (Promega Corporation, catalog number: E2691 )
  14. Opti-MEM Reduced Serum Medium (Life Technologies, catalog number: 31985-062 )
  15. PE conjugated anti-CD 114 (GCSFR) antibody (BD Biosciences, catalog number 554538 )
  16. Cell culture medium (see Recipes)
  17. Sterile FACS buffer (see Recipes)
  18. PEB Buffer (see Recipes)

Equipment

  1. Fluorescence-activated cell sorter (e.g. DAKO Cytomation MoFlo 9-color cell sorter)
  2. 37 °C, 5% CO2 tissue culture incubator
  3. Refrigerated centrifuge
  4. Class 2 biological safety cabinet with laminar flow hood
  5. 70 μm cell strainer (Thermo Fisher Scientific, catalog number: 22-363-548 )
  6. T-75 culture flask or 10 cm dish
  7. Anesthesia machine/chamber with nose cone appropriate for mice (Surgivet or VetEquip)
  8. Fluorescent microscope
  9. Surgical instruments
    1. 5.5-in Mayo-Hegar or similar surgical needle holder (Millennium Surgical or Roboz Surgical Instrument)
    2. Sterile gloves (Thermo Fisher Scientific)
    3. Disposable sterile scalpel blade (#10) (Millennium Surgical or Roboz Surgical Instrument)
    4. 27-G needle  
    5. Sterile 1-cc slip tip syringe
    6. Polysorb 4-0 sutures with RB-1 tapered needle (U.S. Surgical)
    7. 9-mm wound clips  (VWR International)
    8. Rodent ear tags (National Band & Tag Company)

Procedure

Note: All steps must be performed sterilely under tissue culture hood since the cells will be injected into immunodeficient mice.

  1. From a given cell line, establish two lines that express two different fluorescent proteins, e.g. NGP/GFP+ and NGP/tdTomato+.
    Note: Numerous transduction protocols exist.  We used a lentiviral transduction protocol, using FuGENE 6 and Opti-MEM Reduced Serum Medium to transfect 293T cells with the viral packaging plasmids and our construct of interest.  Viral supernatant was collected at 48 and 72 h. Viral supernatant was then used to transduce our neuroblastoma cell line of interest. Cells were incubated with viral supernatant for 24 h and then selected with antibiotic according to the antibiotic resistant gene contained in the plasmid until all non-transduced cells died (4-5 days).
  2. Harvest cells transduced with fluorescent protein using 0.05% Trypsin.
    1. For a T-75, use 1.5 ml Trypsin and 8.5 ml media.
    2. For a 10 cm dish, use 0.5 ml Trypsin and 4.5 ml media.
  3. Transfer to a 15 ml tube and spin down at 250 x g for 5 min.
  4. Resuspend cells in either sterile FACS Buffer or PBS.
    1. Resuspend in 5 ml for a 10 cm dish or 10 ml for a T-75.
  5. Count cells.
    Note: Total number of cells desired depends on how many cells are planned for injection into each mouse and how many mice are being injected for each experiment. For neuroblastoma cell lines, the number of cells injected per mouse can range from 1,000 to 1.0 x 106. For the in vivo lineage-tracing studies performed in Reference 1, 1,000 cells were injected into each mouse.
  6. Label FACS tubes.
  7. Put desired number of cells in FACS tubes and spin down to wash.
    1. Any cells not used for sorting can be put back into culture at this point.
  8. Aspirate the wash. Be very careful not to aspirate the cells. Sometimes they do not adhere as a pellet very well, and they slide around.
    1. If you have to leave a little bit of volume in the tube in order to save the cells, you can add an extra wash to be sure all media/Trypsin has been removed.
  9. Add 2 ml sterile FACS buffer to tubes, vortex, and spin down to wash again. At least 2 washes are necessary.
  10. Aspirate the FACS buffer.
  11. Resuspend cells in FACS buffer in a final concentration of 1 x 107 cells/ml for incubation with primary antibodies. To detect the GCSF-R (CD114), we used PE conjugated anti-CD 114 (GCSFR) antibody. We used 1 μg of antibody per 1.0 x 106 cells in a total volume of 100 µl, however the concentration of antibody varies based on the particular antibody used and the antigen being detected.
    1. For 1 x 106 cells, resuspend in 100 µl total (subtract out the volume of antibody that will be added). If staining more than 1 x 106 cells, you can scale up.
  12. Add antibody, mix well, and immediately keep away from light.
    1. If adding 5 µl of antibody, first resuspend cells in 95 µl of FACS buffer for a total volume of 100 µl.
  13. Incubate cells with antibody for 30 min on ice in the dark.
    1. Can use an ice bucket with a lid in the tissue culture hood.
    2. If staining a large number of cells, it is necessary to vortex them a few times during this incubation because the cells will start to settle to the bottom of the tube.
    3. During this time, put FACS buffer on ice or back in the 4 °C. This should be kept cold.
  14. Add 2 ml FACS buffer to tubes and vortex. Spin down to wash.
  15. Aspirate the wash. Be very careful not to aspirate the cells. Sometimes they do not adhere as a pellet very well, and they slide around.
    1. If you have to leave a little bit of volume in the tube in order to save the cells, you can add an extra wash to be sure all of the extra stain has been removed.
  16. Repeat steps 13-14.
  17. Resuspend cells in FACS buffer for sorting.
    1. For 1 x 106 cells, resuspend in 0.5 ml. You can scale up with a larger number of cells.
  18. Run cells through a 40 µm filter just before sorting so clumps of cells won’t clog the sorting machine.
  19. Sort cells of interest using fluorescence-activated cell sorter, e.g. DAKO Cytomation MoFlo 9-color cell sorter, for example tdTomato+/GCSFR+ cells and GFP+/GCSFR- cells.
  20. Mix cells of differing lineages in desired ratio for injection. For example, NGP/tdTomato+/GCSFR+ cells mixed with NGP/GFP+/GCSFR- cells mixed in a 1:1 ratio.
  21. Inject cells sterilely into immunodeficient mice. For a detailed protocol, see Patterson et al. (2011).
  22. After tumors have grown for desired time period, sacrifice mice and harvest tumors.  
    Note: Time to tumor growth will depend on the number of cells and the cell type injected. If cells have been transduced with a luciferase gene, bioluminescence can be used to monitor tumor growth. Otherwise, mice can be examined and tumors palpated and measured by calipers weekly to detect tumor growth. For an average neuroblastoma cell line, if 1.0 x 106 cells are injected, palpable tumors are typically present by 4 weeks. For more detailed information, please refer to Hsu et al. (2013) and Patterson et al. (2011).
  23. Mice may be sacrificed by CO2 asphyxiation. A necropsy is performed and the tumor resected. Fresh tumors should be kept on ice and in the dark until after gross examination under fluorescent microscope. Tumors should be examined immediately after resection to maintain cell viability.
  24. Tumors may be examined grossly under fluorescent microscope to determine dominant cell lineage, e.g. if tumor is predominately GFP+ or tdTomato+.
  25. To quantify contribution of each lineage to total tumor make-up, prepare tumor cells for flow cytometry.
  26. Place tumor sample immediately in cell culture media (DMEM or RPMI-1640). Mince tumor into very small pieces (approximately 5 mm) using a sterile scalpel blade or sterile scissors in a 10 cm petri dish with a small amount of media.
  27. Gently mechanically disrupt tumor as follows: Place a 70 μm cell strainer in a new petri dish, put small amount of media in dish (enough to coat bottom of dish). In batches, pass media containing tumor sample (from step 1) through cell strainer (gently push tumor through the cell strainer using the back end of 10 ml syringe plunger).
  28. Collect cell suspension in 15 ml tube. Centrifuge. Resuspend in 5 ml media without serum or PBS.
  29. Add 150 μl Collagenase I (10,000 CDU/ml) and 150 μl Dispase II (32 mg/ml) and 2 μl DNase I to solution.
  30. Incubate sample for 20-30 min at 37 °C. Shake or vortex the tube every 10 min during incubation period or place on tube rotator.
  31. Centrifuge sample, aspirate supernatant.
  32. Resuspend the sample in 5 ml of PEB buffer and apply to a cell strainer (70 μm mesh size) placed in a 50 ml tube. Wash the cell strainer with 5 ml PEB buffer.
  33. Discard cell strainer and add PEB buffer to a final volume of 50 ml.
  34. Centrifuge cell suspension at 300 x g for 10 min. Aspirate supernatant completely.
  35. Resuspend cells in FACS buffer to analyze. For 1.0 x 106 cells, resuspend in 0.5 ml of FACS buffer.  
  36. Analyze cells on fluorescence activated flow cytometer to specifically quantify each lineage, i.e. percentage of GFP+ versus tdTomato+ cells in each tumor.

Recipes

  1. Cell culture medium
    Serum-free medium (e.g., RPMI medium 1640)
    Supplemented with 10% (v/v) fetal bovine serum
    1% (v/v) penicillin/streptomycin
    1% (v/v) of 100x L-glutamine (final concentration 2 mM)
  2. Sterile FACS buffer
    Sterile PBS with 1% sterile FBS
    No sodium azide
    Must keep at 4 °C
    Do not keep longer than 1 week
  3. PEB Buffer
    PBS (pH 7.2)
    0.5% BSA
    2 mM EDTA
    Keep buffer cold (2-8 °C)

Acknowledgments

This work was conducted with support from the American Cancer Society (J.M. Shohet), Alex's Lemonade Stand Foundation (J.M. Shohet), Gillson-Longenbaugh Foundation (J.M. Shohet), Children's Neuroblastoma Research Foundation (J.M. Shohet), St. Baldrick's Foundation (E.S. Kim), Texas Children's Hospital Department of Surgery Seed Grant (E.S. Kim), and Texas Children’s Hospital Department of Surgery institutional support (E.S. Kim). We would also like to acknowledge the assistance of the Texas Children’s Cancer Center Flow Cytometry Core.

References

  1. Hsu, D. M., Agarwal, S., Benham, A., Coarfa, C., Trahan, D. N., Chen, Z., Stowers, P. N., Courtney, A. N., Lakoma, A., Barbieri, E., Metelitsa, L. S., Gunaratne, P., Kim, E. S. and Shohet, J. M. (2013). G-CSF receptor positive neuroblastoma subpopulations are enriched in chemotherapy-resistant or relapsed tumors and are highly tumorigenic. Cancer Res 73(13): 4134-4146.
  2. Patterson, D. M., Shohet, J. M. and Kim, E. S. (2011). Preclinical models of pediatric solid tumors (neuroblastoma) and their use in drug discovery. Curr Protoc Pharmacol Chapter 14: Unit 14 17.

简介

肿瘤由异质亚群组成,其可以表现出不同的分化,自我更新和致瘤性能力。 体内谱系追踪研究是定义肿瘤亚群在肿瘤生长中的作用和作为治疗剂靶标的有力工具。 该方案描述了使用用两种不同的荧光蛋白(GFP和tdTomato)转导的神经母细胞瘤癌细胞系来追踪表达GCSF受体(CD114 + sup)+或CD114(sup)+的细胞的特异性作用 )对肿瘤生长的作用。

材料和试剂

  1. 人成神经细胞瘤细胞系(NGP,NB-1691,IMR-32)
    注意:用两种不同的荧光蛋白(例如GFP(Clontech,目录号:632370)和tdTomato(Clontech,目录号:632534))转导细胞系,使得存在GFP阳性线和tdTomato线的相同细胞类型。以这种方式,可以追踪相同细胞类型的亚群(即GCSF-R阳性和GCSF-R阴性细胞)。如果需要通过生物发光成像对肿瘤生长进行体内监测,还应当用商业上可获得的载体转染或病毒转导细胞系。 pGL2-对照载体(Promega Corporation)以表达发光报道基因。关于体内生物发光成像的详细描述和方案,请参考参考文献2.
  2. 4-6周龄雌性非肥胖糖尿病/严重联合免疫缺陷(NOD/SCID)小鼠(Taconic,型号:NODSCF, http://www.taconic.com/NODSC
  3. 293T细胞
  4. RPMI培养基1640(Life Technologies,目录号:11875-101)
  5. 10%(v/v)胎牛血清(FBS)(Life Technologies,目录号:16000044)
  6. 1%(v/v)青霉素/链霉素(Life Technologies,目录号:15140122)(100x P/S;终浓度100单位/ml青霉素和100mg/ml链霉素)
  7. 1%(v/v)100x L-谷氨酰胺(Life Technologies,目录号:25030081)(终浓度2mM)
  8. 磷酸盐缓冲盐水(PBS)(无菌)(Life Technologies,目录号:70011069)
  9. 0.25%胰蛋白酶/EDTA(Life Technologies,目录号:25200056)
  10. 胶原酶I(Sigma-Aldrich,目录号:C0130)[制备在PBS中含有10,000个胶原酶消化单位(CDU/ml)的溶液]
  11. Dispase II(Roche Diagnostics,目录号:04942078001)(制备在PBS中含有32mg/ml Dispase II的溶液)
  12. DNase I(EMD Millipore,Calbiochem ,目录号:260913)(制备含有5MU/ml DNA酶I的溶液)
  13. FuGENE 6(Promega Corporation,目录号:E2691)
  14. Opti-MEM减少血清培养基(Life Technologies,目录号:31985-062)
  15. PE缀合的抗CD114(GCSFR)抗体(BD Biosciences,目录号554538)
  16. 细胞培养基(参见配方)
  17. 无菌FACS缓冲液(参见配方)
  18. PEB缓冲区(参见配方)

设备

  1. 荧光激活细胞分选仪(例如DAKO Cytomation MoFlo 9色细胞分选仪)
  2. 37℃,5%CO 2组织培养箱中培养
  3. 冷冻离心机
  4. 具有层流罩的2级生物安全柜
  5. 70μm细胞过滤器(Thermo Fisher Scientific,目录号:22-363-548)
  6. T-75培养瓶或10cm培养皿
  7. 具有适合于小鼠(Surgivet或VetEquip)的鼻锥的麻醉机/腔室
  8. 荧光显微镜
  9. 手术器械
    1. 5.5在Mayo-Hegar或类似的手术针支架(Millennium Surgical或Roboz外科手术器械)中
    2. 无菌手套(Thermo Fisher Scientific)
    3. 一次性无菌手术刀刀片(#10)(Millennium Surgical或Roboz外科手术器械)
    4. 27-G针
    5. 无菌1-cc滑动注射器
    6. Polysorb 4-0缝合用RB-1锥形针(美国外科)
    7. 9毫米伤口夹 (VWR国际)
    8. 啮齿动物耳标(National Band& Tag Company)

程序

注意:所有步骤必须在组织培养罩下无菌地进行,因为细胞将被注射到免疫缺陷小鼠中。

  1. 从给定的细胞系,建立两条表达两种不同荧光蛋白的细胞系,例如 NGP/GFP + 和NGP/tdTomato + /> 注意:存在大量的传导协议。我们使用慢病毒转导方案,使用FuGENE 6和Opti-MEM还原血清培养基,用病毒包装质粒和我们的感兴趣的构建体转染293T细胞。在48和72小时收集病毒上清液。然后使用病毒上清液转导我们的感兴趣的成神经细胞瘤细胞系。将细胞与病毒上清液孵育24小时,然后根据质粒中包含的抗生素抗性基因用抗生素选择,直到所有非转导细胞死亡(4-5天)。
  2. 收获细胞用荧光蛋白使用0.05%胰蛋白酶转导。
    1. 对于T-75,使用1.5ml胰蛋白酶和8.5ml培养基
    2. 对于10cm培养皿,使用0.5ml胰蛋白酶和4.5ml培养基。
  3. 转移到15ml管中并在250×g下离心5分钟
  4. 重悬细胞在无菌FACS缓冲液或PBS。
    1. 对于10cm培养皿重悬于5ml,或对于T-75重悬于10ml。
  5. 计数单元格。
    注意:所需的细胞总数取决于计划注射到每只小鼠中的细胞数量,以及每次实验注射多少小鼠。 对于成神经细胞瘤细胞系,每只小鼠注射的细胞数目可以为1,000至1.0×10 10个 6 。 对于参考文献1中进行的体内谱系追踪研究,向每只小鼠注射1,000个细胞。
  6. 标签FACS管
  7. 将所需数量的细胞在FACS管中,旋转以洗涤。
    1. 任何不用于分选的细胞可以在这一点上回到培养中。
  8. 吸出洗液。 要非常小心,不要吸出细胞。 有时候,他们不会坚持作为一个丸很好,他们滑了。
    1. 如果你要留一点点的音量在管中,以保存   细胞,你可以添加一个额外的洗,以确保所有的媒体/胰蛋白酶 已删除。
  9. 加入2毫升无菌FACS缓冲液的试管,涡旋,旋转下来再次洗涤。 至少需要洗2次。
  10. 吸出FACS缓冲液。
  11. 重悬细胞在FACS缓冲液中,终浓度为1×10 7个细胞/ml,用于与一抗孵育。 为了检测GCSF-R(CD114),我们使用PE缀合的抗CD114(GCSFR)抗体。 我们使用1μ g抗体/1.0×10 6个细胞,总体积为100μl,然而抗体的浓度根据所用的特定抗体和检测的抗原而变化。
    1. 对于1×10 6个细胞,重悬于总共100μl(减去体积 的抗体)。 如果染色超过1×10 6个细胞, 你可以放大。
  12. 加入抗体,混匀,立即避光。
    1. 如果加入5μl抗体,首先将细胞重悬于95μlFACS缓冲液中,总体积为100μl。
  13. 孵育细胞与抗体在冰上在黑暗中30分钟。
    1. 可以在组织培养罩中使用带盖的冰桶。
    2. 如果染色大量的细胞,有必要涡旋它们 在该孵育期间几次,因为细胞将开始沉降 到管的底部
    3. 在此期间,将FACS缓冲液置于冰上或放回4℃。 这应该保持冷。
  14. 向管中加入2ml FACS缓冲液并涡旋。 旋下来洗。
  15. 吸出洗液。 要非常小心,不要吸出细胞。 有时候,他们不会坚持作为一个丸很好,他们滑了。
    1. 如果你要留一点点的音量在管中,以保存   细胞,你可以添加额外的洗涤,以确保所有的额外染色 已被删除。
  16. 重复步骤13-14。
  17. 重悬细胞在FACS缓冲液中进行分选。
    1. 对于1×10 6个细胞,重悬于0.5ml。 您可以使用更大数量的单元格进行扩展。
  18. 在分选之前,通过40μm过滤器运行细胞,因此细胞团不会堵塞分选机
  19. 使用荧光激活细胞分选仪,例如 DAKO Cytomation MoFlo 9色细胞分选仪,例如tdTomato + /GCSFR + 细胞,和GFP + /GCSFR - 细胞
  20. 以所需比例混合不同谱系的细胞用于注射。例如,将与NGP/GFP混合的NGP/tdTomato + /GCSFR + 细胞与/GCSFR 以1:1的比例
  21. 注射细胞无菌地进入免疫缺陷小鼠。有关详细协议,请参阅Patterson等人(2011)。
  22. 在肿瘤生长期望的时间后,处死小鼠并收获肿瘤。  
    注意:肿瘤生长的时间将取决于细胞的数量和注射的细胞类型。如果细胞已经用荧光素酶基因转导,生物发光可以用于监测肿瘤生长。否则,可以检查小鼠并且每周用卡尺测量肿瘤并检测肿瘤生长。对于平均神经母细胞瘤细胞系,如果注射1.0×10 6个细胞,则可触及的肿瘤通常存在4周。更多详细信息,请参考Hsu et al。 (2013)和Patterson et al。 (2011)。
  23. 小鼠可以通过CO 2窒息处死。进行尸检并切除肿瘤。新鲜的肿瘤应该保存在冰和黑暗中,直到在荧光显微镜下的总检查。切除后应立即检查肿瘤,以维持细胞活力
  24. 如果肿瘤主要是GFP + 或tdTomato + ,可以在荧光显微镜下粗略检查肿瘤以确定优势细胞谱系。例如。
  25. 为了量化每个谱系对总肿瘤组成的贡献,制备用于流式细胞术的肿瘤细胞
  26. 将肿瘤样品立即在细胞培养基(DMEM或RPMI-1640)。使用无菌手术刀或无菌剪刀在具有少量培养基的10cm陪替氏培养皿中将ince肿瘤切成非常小的块(约5mm)。
  27. 轻轻地机械破坏肿瘤如下:将一个70微米的细胞过滤器在一个新的培养皿中,放入少量的培养基在培养皿中(足够涂在培养皿的底部)。分批,将含有肿瘤样品(来自步骤1)的培养基通过细胞过滤器(使用10ml注射器柱塞的后端轻轻推动肿瘤通过细胞过滤器)。
  28. 收集细胞悬浮在15毫升管中。 离心机。 重悬于5ml无血清或PBS的培养基中
  29. 向溶液中加入150μl胶原酶I(10,000CDU/ml)和150μlDispase II(32mg/ml)和2μlDNA酶I。
  30. 在37℃下孵育样品20-30分钟。 在孵育期间每10分钟振荡或涡旋管,或置于管旋转器上
  31. 离心样品,吸出上清液
  32. 将样品重悬于5ml PEB缓冲液中,并施加于置于50ml管中的细胞滤器(70μm目尺寸)。 用5 ml PEB缓冲液洗涤细胞滤器。
  33. 弃去细胞过滤器,加入PEB缓冲液至终体积为50ml
  34. 在300×g离心细胞悬浮液10分钟。 完全吸出上清液
  35. 重悬细胞在FACS缓冲液中进行分析。 对于1.0×10 6个细胞,重悬于0.5ml的FACS缓冲液中。  
  36. 在荧光激活的流式细胞仪上分析细胞以特异性地定量每个谱系,即每个肿瘤中GFP + 对tdTomato + 细胞的百分比。 >

食谱

  1. 细胞培养基
    无血清培养基(例如,,RPMI培养基1640)
    补充10%(v/v)胎牛血清 1%(v/v)青霉素/链霉素 1%(v/v)100×L-谷氨酰胺(终浓度2mM)
  2. 无菌FACS缓冲区
    含1%无菌FBS的无菌PBS
    无叠氮化钠
    必须保持在4°C
    不要超过1周
  3. PEB缓冲区
    PBS(pH 7.2)
    0.5%BSA
    2mM EDTA 保持缓冲液冷(2-8°C)

致谢

这项工作是在美国癌症协会(JM Shohet),Alex的柠檬水支架基金会(JM Shohet),Gillson-Longenbaugh基金会(JM Shohet),儿童神经母细胞瘤研究基金会(JM Shohet),圣巴德里克基金会),德克萨斯儿童医院外科种子授粉(ES Kim)和德克萨斯儿童医院外科系统支持(ES Kim)。我们还要感谢德克萨斯儿童癌症中心流式细胞计数核心的帮助。

参考文献

  1. H,DM,Agarwal,S.,Benham,A.,Coarfa,C.,Trahan,DN,Chen,Z.,Stowers,PN,Courtney,AN,Lakoma,A.,Barbieri,E.,Metelitsa, Gunaratne,P.,Kim,ES和Shohet,JM(2013)。 G-CSF受体阳性神经母细胞瘤亚群富含化疗耐药或复发性肿瘤,并且具有高度致瘤性。 Cancer Res 73(13):4134-4146。
  2. Patterson,D.M.,Shohet,J.M。和Kim,E.S。(2011)。 儿科实体瘤(神经母细胞瘤)的临床前模型及其在药物发现中的应用 < em> Curr Protoc Pharmacol Chapter 14:Unit 14 17.
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Hsu (Maiden name: Danielle M. Patterson), D. M., Shohet, J. M. and Kim, E. S. (2014). In vivo Lineage-tracing Studies in a Cancer Stem Cell Population in Neuroblastoma. Bio-protocol 4(8): e1104. DOI: 10.21769/BioProtoc.1104.
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