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In vitro and in vivo Limiting Dilution Assay for Colorectal Cancer
结肠直肠癌的体外和体内限制稀释测定法   

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Abstract

The in vitro limiting dilution assay is used to determine the colorectal cancer initiating cell (CC-IC) frequency of a CC-IC enriched suspension culture, grown in growth factor enriched serum free media. The in vivo limiting dilution assay is used to determine the colorectal cancer initiating cell frequency of a primary colorectal cancer sample or an established suspension cell line using immunocompromised murine xenograft models. In vitro and in vivo limiting dilution assays (LDAs) can be used to determine the effect of a specific treatment or genetic knockdown strategy on the initiating cell frequency of a population of CC-ICs or colorectal cancer sample, respectively.

Part I. In vitro CRC LDA

Materials and Reagents

  1. Centrifuge tubes (BD Falcon, catalog numbers: 352096 and 352070 )
    Note: Currently, it is “Corning Inc., FalconTM, catalog numbers: 352096 and 352070”.
  2. 7x sterile, non-tissue culture 96 well U-bottom plates (Corning Inc., FalconTM, catalog number: 351177 )
  3. FACS tubes with 0.35 μm filter (BD Falcon, catalog number: 352235 )
    Note: Currently, it is “Corning Inc., FalconTM, catalog number: 352235”.
  4. Wash media (DMEM/F-12) (Thermo Fisher Scientific, GibcoTM, catalog number: 11320-033 )
  5. 0.4% Trypan blue solution (Life Technologies, catalog number: 15250-061 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 15250-061”.
  6. Permanent marker (VWR International, catalog number: 52877-310 )
  7. 0.25% Trypsin (Life Technologies, catalog number: 25200-056 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 25200-056”.
  8. Penicillin-streptomycin-amphotericin B (1%) (Life Technologies, catalog number: 15240-062 )
    Note: Currently, it is “Antibiotic-Antimycotic (100x) (Thermo Fisher Scientific, GibcoTM, catalog number: 15240-062)".
  9. L-glutamine (2 mM) (Invitrogen, catalog number: 25030081 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 25030081”.
  10. Non-essential amino acids (1x) (Thermo Fisher Scientific, HycloneTM, catalog number: SH3023801 )
  11. Sodium pyruvate (1 mM) (Thermo Fisher Scientific, HycloneTM, catalog number: SH3023901 )
  12. HEPES (1x) (Invitrogen, catalog number: 15630080 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 15630080”.
  13. Heparin sodium salt (4 μg/ml) from porcine intestinal mucosa (Sigma-Aldrich, catalog number: H3149-100KU )
  14. Lipids (Sigma-Aldrich, catalog number: L0288 )
  15. Human Epidermal Growth Factor Recombinant (EGF) (20 ng/ml) (Biomartcanada, catalog number: RKP01133 )
  16. Human basic Fibroblast Growth Factor Recombinant (bFGF) (10 ng/ml) (Biomartcanada, catalog number: RKP09038 )
  17. N2 supplement-A (STEMCELL Technologies, catalog number: 07152 )
  18. NeuroCult SM1 Neuronal (STEMCELL Technologies, catalog number: 05711 )
  19. Sytox blue (Life Technologies, catalog number: S11348 )
    Note: Currently, it is “Thermo Fisher Scientific, Molecular ProbesTM, catalog number: S11348”.
  20. Phosphate-buffered saline (PBS) (Life Technologies, catalog number: 10010023 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 10010023”.
  21. 2% fetal bovine serum (FBS) (Life Technologies, catalog number: 26140-111 )
    Note: Currently, it is “Thermo Fisher Scientific, Gibco™, catalog number: 26140-111 ”.
  22. Serum free cell culture media (see Recipes)

Equipment

  1. 37 °C Incubator
  2. Light microscope (Leica Microsystems, model: DM IL LED )
  3. Hemocytometer (Hausser Scientific, catalog number: 1483 )
  4. Pipette aid (INTEGRA Biosciences AG, catalog number: 155 000 )
  5. Pipettes (BD Falcon, catalog number: 357530 )
    Note: Currently, it is “Corning Inc., FalconTM, catalog number: 357530”.
  6. Flow cytometry assisted cell sorter (FACS)

Procedure

Note: The range of cell concentrations used will depend on the frequency of initiating cells in your fraction. An initial LDA using a wide range of concentrations can be used to find the best range of cell doses, ideally it should include doses with 100% sphere formation down to cell doses with no sphere formation, as well as, a range of doses in between. For an LDA to be statistically valid one must first test whether their sample complies with the Poisson single-hit model, which assumes that the number of biologically active units in a culture varies according to Poisson distribution. For additional information and calculations please refer to Extreme Limiting Dilution Analysis (ELDA) site (http://bioinf.wehi.edu.au/software/elda/), and the accompanying paper (Hu and Smyt, 2009). Ex: if the initiating frequency is approximately (1:50-1:200), then the range indicated in Table 1 will be sufficient to obtain statistically significant results.

  1. Seeding for an in vitro LDA
    1. Grow cells to confluence in a T175 flask at 37 °C.
    2. Centrifuge cells (300-450 x g) for 5 min at room temperature, then remove supernatant.
    3. Resuspend cell pellet in ~3 ml of trypsin and incubate at 37 °C for the necessary time to obtain single cells.
    4. Remove cells from incubator and resuspend with ~10 ml of wash media to stop trypsin reaction.
    5. Centrifuge cells (300-450 x g) for 5 min at room temperature, then remove supernatant.
    6. Completely resuspend pellet in enough cell media so that you will have a final cell concentration of about 1 x 106 cells/ml in order to make cell counting on the hemocytometer easier for you.
    7. Mix 10 μl of cell mixture with 10 μl of trypan blue solution and load mixture onto a hemocytometer to count the number of live cells using a light microscope. Be sure to count cells in all four coordinates and divide by 2 to obtain the concentration of cells in your solution (__x 104/ml). Please refer to http://www.abcam.com/protocols/counting-cells-using-a-haemocytometer for a further explanation on how to use a hemocytometer.
    8. Seed cells either manually or by using a FACS machine.

      Manual cell seeding
      1. Serially dilute cells to obtain the following final cell concentrations
        1. Tube 1 (1,000 cells/well): 27,000 cells in 5.4 ml media
        2. Tube 2 (100 cells/well): 10,000 cells in 20 ml media
        3. Tube 3 (10 cells/well): 2,000 cells in 40 ml media
        4. Tube 4 (1 cell/well): 300 cells in 60 ml media
      2. Thoroughly mix cell solutions each time, before dispensing 200 μl of each into sterile non-tissue culture 96 well U-bottom plates according to Table 1.
      3. Centrifuge plates containing single cells (300-450 x g) for 5 min at room temperature, and using a microscope, determine which wells are confirmed to have only one cell. Using a marker, circle those wells containing single cells.
      4. Return plates to 37 °C incubator as soon as possible.

        Table 1. Cell seeding for an in vitro limiting dilution assay
        Cell concentration (#cells/well)
        Number of 96 well plates
        Number of wells
        1,000
        0.25
        24
        100
        1
        96
        10
        2
        192
        1
        3
        288

      Flow cytometry assisted cell sorter (FACS) cell seeding
      1. Remove ~2-3 x 106 cells from your cell solution and put it into a centrifuge.
      2. Centrifuge cells (300-450 x g) for 5 min at room temperature.
      3. While cells are spinning, mix sytox blue with PBS + 2% FBS to obtain a final working concentration of 10 nM-1 μM depending on your cell line (please refer to product manual for further information). Avoid exposure of sytox blue solution to light.
      4. Remove supernatant and completely resuspend pellet in 2-3 ml sytox blue working solution.
      5. Filter cell/sytox blue solution through a 0.35 μm filter to ensure a single cell suspension before putting cells into a FACS tube.
      6. Keep the cell solution on ice and in the dark.
      7. Add 200 μl of cell media to 7 sterile non-tissue culture 96 well U-bottom plates.
      8. Use a FACS machine to sort cells into 96 well plates containing cell media, gating out the dead cells that are positive for sytox blue. Sytox blue has an excitation of 444 nm and an emission of 480 nm (please refer to product manual for further information on how to read sytox blue on a FACS machine).
      9. Centrifuge plates containing single cells (300-450 x g) for 5 min at room temperature, and using a microscope, determine which wells are confirmed to have only one cell. Using a marker, circle those wells containing single cells.
      10. Return plates to 37 °C incubator as soon as possible.


        Figure 1. In vitro LDA schematic

  2. Scoring an in vitro LDA
    1. After ~2-4 weeks remove the plates from the incubator and using a microscope, determine which wells contain cell spheres. For the 1 cell/well plates, be sure to only score those wells that were originally confirmed to contain a single cell.
    2. Use ELDA: Extreme Limiting Dilution Analysis online software to calculate the cancer cell initiating frequency and significance (http://bioinf.wehi.edu.au/software/elda/).

Part II. In vivo LDA

Materials and Reagents

  1. Centrifuge tubes (BD Falcon, catalog numbers: 352096 and 352070 )
    Note: Currently, it is “Corning Inc., FalconTM, catalog numbers: 352096 and 352070”.
  2. 1 ml syringe without needle (Restek Corporation, catalog number: 22766)
  3. 100 x 1 ml syringes with (281/2G) needles (BD, catalog number: 329420 )
  4. 50x NOD-SCID or NSG mice
  5. ~5 ml matrigel (Corning, catalog number: 354248 )
  6. Wash media (DMEM/F-12) (Thermo Fisher Scientific, GibcoTM, catalog number: 11320-033 )
  7. 0.4% Trypan blue solution (Life Technologies, catalog number: 15250-061 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 15250-061”.
  8. 0.25% Trypsin (Life Technologies, catalog number: 25200-056 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 25200-056”.
  9. Penicillin-streptomycin-amphotericin B (1%) (Life Technologies, catalog number: 15240-062 )
    Note: Currently, it is “Antibiotic-Antimycotic (100x) (Thermo Fisher Scientific, GibcoTM, catalog number: 15240-062)".
  10. L-glutamine (2 mM) (Invitrogen, catalog number: 25030081 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 25030081”.
  11. Non-essential amino acids (1x) (Thermo Fisher Scientific, HycloneTM, catalog number: SH3023801 )
  12. Sodium pyruvate (1 mM) (Thermo Fisher Scientific, HycloneTM, catalog number: SH3023901 )
  13. HEPES (1x) (Invitrogen, catalog number: 15630080 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 15630080”.
  14. Heparin sodium salt (4 μg/ml) from porcine intestinal mucosa (Sigma-Aldrich, catalog number: H3149-100KU )
  15. Lipids (Sigma-Aldrich, catalog number: L0288 )
  16. Human Epidermal Growth Factor Recombinant (EGF) (20 ng/ml) (Biomartcanada, catalog number: RKP01133 )
  17. Human basic Fibroblast Growth Factor Recombinant (bFGF) (10 ng/ml) (Biomartcanada, catalog number: RKP09038 )
  18. N2 supplement-A (STEMCELL Technologies, catalog number: 07152 )
  19. NeuroCult SM1 Neuronal (STEMCELL Technologies, catalog number: 05711 )
  20. Serum free cell culture media (see Recipes)

Equipment

  1. 37 °C Incubator
  2. Light microscope (Leica Microsystems, model: DM IL LED )
  3. Hemocytometer (Hausser Scientific, catalog number: 1483 )
  4. Pipette aid (INTEGRA Biosciences AG, catalog number: 155000 )
  5. Pipettes (BD Falcon, catalog number: 357530 )
    Note: Currently, it is “Corning Inc., FalconTM, catalog number: 357530”.
  6. Mouse ear punch (Kent Scientific Corporation, catalog number: INS750075-5)

Procedure

Note: The range of cell concentrations used will depend on the frequency of initiating cells in your fraction. An initial LDA using a wide range of concentrations can be used to find the best range of cell doses, ideally it should include doses with 100% tumor formation down to cell doses with no tumor formation, as well as, a range of doses in between. For an LDA to be statistically valid one must first test whether their sample complies with the Poisson single-hit model, which assumes that the number of biologically active units in a culture varies according to Poisson distribution. For additional information and calculations please refer to ELDA site (http://bioinf.wehi.edu.au/software/elda/), and the accompanying paper (Hu and Smyt, 2009). Ex: if the initiating frequency is approximately (1:1,000-1:5,000), then the range indicated in Table 2 will be sufficient to obtain statistically significant results.

  1. Preparing cell syringes
    1. Grow cells to confluence in a T175 flask at 37 °C.
    2. Centrifuge cells (300-450 x g) for 5 min at room temperature, then remove supernatant.
    3. While cells are spinning, thaw matrigel on ice.
    4. Resuspend pellet in ~3 ml of trypsin and incubate at 37 °C for the necessary time to obtain single cells.
    5. Remove cells from incubator and resuspend with ~10 ml of wash media to stop trypsin reaction.
    6. Centrifuge cells (300-450 x g) for 5 min at room temperature, then remove supernatant
    7. Completely resuspend pellet in enough cell media so that you will have a final cell concentration of about 3 x 106 cells/ml in order to make cell counting on the hemocytometer easier for you. Ensure that your cell concentration is no less than 2 x 106 cells/ml to allow for the cell concentrations listed in step A9.
    8. Mix 10 μl of cell mixture with 10 μl of trypan blue and load mixture onto a hemocytometer to count the number of live cells using a light microscope. Be sure to count cells in all four coordinates and divide by 2 to obtain the concentration of cells in your solution (__ x 104/ml). Please refer to http://www.abcam.com/protocols/counting-cells-using-a-haemocytometer for a further explanation on how to use a hemocytometer.
    9. Serially dilute cells so that you have the following final cell concentrations.
      1. Tube 1 (50,000 cells/injection): 600,000 cells in 360 μl media
      2. Tube 2 (10,000 cells/injection): 120,000 cells in 360 μl media
      3. Tube 3 (1,000 cells/ injection): 12,000 cells in 360 μl media
      4. Tube 4 (100 cells/injection): 1200 cells in 360 μl media
      5. Tube 5 (10 cells/injection): 120 cells in 360 μl media
    10. Prepare syringes (with needles) by removing the plunger and adding necessary labels (i.e., cell concentration, treatment etc.).
    11. Using a syringe (without a needle), aspirate the matrigel and deposit a small drop of gel (~200 μl) into the base of syringe.
    12. Thoroughly mix the desired cell solution before adding 30 μl to the droplet of matrigel.
    13. Carefully reinsert the plunger, making sure not to expel the matrigel solution out of the syringe.
    14. Keep the loaded syringes on ice until ready to use.

      Table 2. Cell concentrations and injections for an in vivo limiting dilution assay
      Cell concentration
      (#cells/injection)
      Number of injections
      (2 injections/mouse)
      Number of mice
      50,000
      20
      10
      10,000
      20
      10
      1,000
      20
      10
      100
      20
      10
      10
      20
      10
      Total:
      100
      50

    15. Repeat this until you have 100 syringes (20 syringes per cell concentration), as shown in Table 2.

  2. Injecting mice
    1. Lightly sedate mice with isoflurane and ear notch them to identify each mouse (see Laboratory Animal Medicine, 2015, for more information on animal identification).
    2. Inject mice subcutaneously with cells on both the right and left flank, using a new syringe for each injection (see Figure 2). Each mouse should have two injections, one on each flank (see Table 2).
    3. Return mice to their cage to recover.


      Figure 2. Subcutaneous injection sites on the right and left flank of a mouse


      Figure 3. In vivo LDA schematic

  3. Scoring an in vivo LDA
    1. After ~4-8 weeks, determine which mice have developed tumors.
    2. Continue to monitor mice with 0-1 tumors for up to 6 months, or as long as ethically permitted, to ensure there is no late tumor formation.
    3. Use ELDA: Extreme Limiting Dilution Analysis online software to calculate the cancer cell initiating frequency and significance (http://bioinf.wehi.edu.au/software/elda/).

Notes

  1. Results will vary slightly when repeating an LDA, however when comparing treated groups, the LDA results should convey the same overall result each time. For example; treatment ‘A’ always produces a lower, statistically significant, initiating frequency compared to a control treatment ‘B’.
  2. Increasing the number of replicates in both in vitro and in vivo LDAs will increase the preciseness of the assay.

Recipes

  1. Serum free cell culture medium
    DMEM/F-12 (1:1 ratio) supplemented with:
    1. Penicillin-streptomycin-amphotericin B (1%) 5 ml/500 ml
    2. L-glutamine (2 mM) 5 ml/500 ml
    3. Non-essential amino acids (1x) 5 ml/500 ml
    4. Sodium pyruvate (1 mM) 5 ml/500 ml
    5. HEPES (1x) 5 ml/500 ml
    6. Heparin sodium salt (4 μg/ml) from porcine intestinal mucosa
    7. Lipids 1 ml/500 ml
    8. Human Epidermal Growth Factor Recombinant (EGF) (20 ng/ml)
    9. Human basic Fibroblast Growth Factor Recombinant (bFGF) (10 ng/ml)
    10. N2 supplement-A 5 ml/500 ml
    11. NeuroCult SM1 Neuronal 2 ml/500 ml

References

  1. Counting cells using a hemocytometer: Abcam.
  2. Fazekas de St, G. (1982). The evaluation of limiting dilution assays. J Immunol Methods 49(2): R11-23.
  3. Hu, Y. and Smyth, G. K. (2009). ELDA: extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays. J Immunol Methods 347(1-2): 70-78.
  4. Kreso, A., van Galen, P., Pedley, N. M., Lima-Fernandes, E., Frelin, C., Davis, T., Cao, L., Baiazitov, R., Du, W., Sydorenko, N., Moon, Y. C., Gibson, L., Wang, Y., Leung, C., Iscove, N. N., Arrowsmith, C. H., Szentgyorgyi, E., Gallinger, S., Dick, J. E. and O'Brien, C. A. (2014). Self-renewal as a therapeutic target in human colorectal cancer. Nat Med 20(1): 29-36.
  5. Strijbosch, L. W., Buurman, W. A., Does, R. J., Zinken, P. H. and Groenewegen, G. (1987). Limiting dilution assays. Experimental design and statistical analysis. J Immunol Methods 97(1): 133-140.
  6. Talcott, M., Akers, W., and Marini, R. (2015). Techniques of experimentation. In: Anderson, L. and Otto, G. (eds). Laboratory Animal Medicine. Elsevier, 1202-1204.

简介

体外极限稀释测定法用于测定在富含生长因子的无血清培养基中生长的CC-IC富集悬浮培养物的结肠直肠癌起始细胞(CC-IC)频率。 体内限制性稀释测定法用于使用免疫受损的鼠异种移植物模型确定原发性结肠直肠癌样品或建立的悬浮细胞系的结肠直肠癌起始细胞频率。 可以使用体外和体内有限稀释测定法(LDAs)来确定特定处理或遗传击倒策略对CC- IC或结肠直肠癌样品。

第I部分。体外 CRC LDA

材料和试剂

  1. 离心管(BD Falcon,目录号:352096和352070)
    ,目录号:352096和352070"。
  2. 7x无菌,非组织培养96孔U型底板(Corning Inc.,Falcon ,目录号:351177)。
  3. 具有0.35μM过滤器(BD Falcon,目录号:352235)的FACS管
    TM ,目录号:352235"。
  4. 洗涤介质(DMEM/F-12)(Thermo Fisher Scientific,Gibco TM ,目录号:11320-033)
  5. 0.4%台盼蓝溶液(Life Technologies,目录号:15250-061)
    注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:15250-061"。
  6. 永久标记(VWR International,目录号:52877-310)
  7. 0.25%胰蛋白酶(Life Technologies,目录号:25200-056) 注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:25200-056"
  8. 青霉素 - 链霉素 - 两性霉素B(1%)(Life technologies,目录号:15240-062)
    注意: 目前,它是"抗生素 - 抗真菌剂(100x)(Thermo Fisher Scientific,Gibco TM >,目录号:15240-062)"。
  9. L-谷氨酰胺(2mM)(Invitrogen,目录号:25030081) 注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:25030081" />
  10. 非必需氨基酸(1x)(Thermo Fisher Scientific,Hyclone TM ,目录号:SH3023801)
  11. 丙酮酸钠(1mM)(Thermo Fisher Scientific,Hyclone TM,目录号:SH3023901)
  12. HEPES(1x)(Invitrogen,目录号:15630080) 注意:目前,"赛默飞世尔科技,Gibco TM ,目录号:15630080" />
  13. 来自猪肠粘膜的肝素钠盐(4μg/ml)(Sigma-Aldrich,目录号:H3149-100KU)
  14. 脂质(Sigma-Aldrich,目录号:L0288)
  15. 人表皮生长因子重组体(EGF)(20ng/ml)(Biomartcanada,目录号:RKP01133)
  16. 人碱性成纤维细胞生长因子重组体(bFGF)(10ng/ml)(Biomartcanada,目录号:RKP09038)
  17. N 2补充物(STEMCELL Technologies,目录号:07152)
  18. NeuroCult SM1神经元(STEMCELL Technologies,目录号:05711)
  19. Sytox blue(Life Technologies,目录号:S11348)
    注意:目前,"Thermo Fisher Scientific,Molecular Probes TM ,目录号:S11348" br />
  20. 磷酸盐缓冲盐水(PBS)(Life Technologies,目录号:10010023)
    注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:10010023" />
  21. 2%胎牛血清(FBS)(Life Technologies,目录号:26140-111) 注意:目前,它是"Thermo Fisher Scientific,Gibco TM,目录号:26140-111"。
  22. 无血清细胞培养基(参见配方)

设备

  1. 37°C孵育器
  2. 光学显微镜(Leica Microsystems,型号:DM IL LED)
  3. 血球计(Hausser Scientific,目录号:1483)
  4. 吸移助剂(INTEGRA Biosciences AG,??目录号:155000)
  5. 移液管(BD Falcon,目录号:357530)
    注意:目前,"Corning Inc.,Falcon TM ,目录号:357530"。
  6. 流式细胞仪辅助细胞分选仪(FACS)

程序

注意:使用的细胞浓度范围将取决于您的部分中起始细胞的频率。使用宽范围浓度的初始LDA可以用于找到最佳的细胞剂量范围,理想地,其应当包括具有100%球形成直到没有球形成的细胞剂量的剂量,以及其间的剂量范围。为了使LDA在统计学上有效,必须首先测试它们的样品是否符合泊松单一命中模型,其假定培养物中的生物活性单元的数目根据泊松分布而变化。有关添加信息和计算,请参考极限稀释分析(ELDA)网站(http://bioinf.wehi.edu.au/software/elda/)和随附的文章(Hu和Smyt,2009)。例如:如果启动频率约为(1:50-1:200),则表1所示的范围将足以获得统计上显着的结果。

  1. 在体外接种 LDA
    1. 在37℃下在T175烧瓶中培养细胞汇合
    2. 在室温下离心细胞(300-450×g)5分钟,然后除去上清液。
    3. 重悬细胞沉淀在?3毫升的胰蛋白酶,孵育在37°C必要的时间,以获得单个细胞。
    4. 从培养箱中取出细胞,并用?10ml的洗涤培养基重悬,以停止胰蛋白酶反应。
    5. 在室温下离心细胞(300-450×g)5分钟,然后除去上清液。
    6. 完全重悬颗粒在足够的细胞培养基,以便你会 具有约1×10 6个细胞/ml的最终细胞浓度 使血细胞计数器上的细胞计数更容易。
    7. 混合10μl ?的细胞混合物与10μl台盼蓝溶液和负载混合物 到血细胞计数器上以使用光计数活细胞的数量 显微镜。确保计算所有四个坐标中的单元格并除以 ?2以获得溶液中细胞的浓度(__×10 4/sup/ml)。 请参阅 http://www.abcam.com/protocols/counting-细胞使用a-血细胞计数器 ?关于如何使用血细胞计数器的进一步解释。
    8. 种子细胞手动或通过使用FACS机。

      手动细胞接种
      1. 连续稀释细胞以获得以下最终细胞浓度
        1. 管1(1,000细胞/孔):在5.4ml培养基中的27,000个细胞
        2. 管2(100细胞/孔):在20ml培养基中的10,000个细胞
        3. 管3(10个细胞/孔):在40ml培养基中的2,000个细胞
        4. 管4(1个细胞/孔):在60ml培养基中有300个细胞
      2. 每次充分混合细胞溶液,在分配200μl之前 ?每个进入无菌非组织培养96孔U型底板 到表1.
      3. 离心板含有单细胞(300-450× ?g)在室温下孵育5分钟,并使用显微镜确定 这些孔被证实仅具有一个细胞。使用标记,圆 那些含有单细胞的孔。
      4. 尽快将培养板放回37°C培养箱
        表1.用于体外有限稀释测定的细胞接种
        细胞浓度(#细胞/孔)
        96孔板的数量
        孔数
        1,000
        0.25
        24
        100
        1
        96
        10
        2
        192
        1
        3
        288

      流式细胞术辅助细胞分选(FACS)细胞接种
      1. 从细胞溶液中取出约2-3×10 6个细胞,并将其放入离心机中
      2. 在室温下离心细胞(300-450×g)5分钟。
      3. 当细胞旋转时,将sytox蓝与PBS + 2%FBS混合以获得 ?最终工作浓度为10nM -1 μM,取决于您的细胞系 ?(有关详细信息,请参阅产品手册)。避免 曝光的蓝色溶液的光。
      4. 去除上清液,并完全重悬在2-3毫升sytox蓝色工作溶液中的沉淀。
      5. 通过0.35μM过滤器过滤细胞/sytox蓝色溶液,以确保a ?单细胞悬浮液,然后将细胞放入FACS管中
      6. 保持细胞溶液在冰和黑暗中。
      7. 加入200微升细胞培养基到7个无菌非组织培养96孔U型底板。
      8. 使用FACS机将细胞分选到含有细胞的96孔板中 ?介质,门控出对sytox蓝色呈阳性的死细胞。 Sytox蓝具有444nm的激发和480的发射 nm(有关如何进一步信息,请参阅产品手册 在FACS机器上读取sytox蓝色)。
      9. 离心板 含有单细胞(300-450×g)在室温下孵育5分钟, ?使用显微镜,确定哪些孔确认只有 一个单元格。使用标记,圈出那些含有单个细胞的孔。
      10. 尽快将培养板放回37°C培养箱

        图1.

  2. 在体外锻炼 LDA
    1. 在?2-4周后,从培养箱中取出板,并使用a 显微镜,确定哪些孔含有细胞球。对于1 细胞/孔板,一定要只对原来的那些孔评分 ?证实含有单细胞。
    2. 使用ELDA:极限 稀释分析在线软件计算癌细胞 启动频率和意义 ( http://bioinf.wehi.edu.au/software/elda/) 。

      表1.用于体外有限稀释测定的细胞接种
      细胞浓度(#细胞/孔)
      96孔板的数量
      孔数
      1,000
      0.25
      24
      100
      1
      96
      10
      2
      192
      1
      3
      288

第II部分 LDA

材料和试剂

  1. 离心管(BD Falcon,目录号:352096和352070)
    注意:目前,"Corning Inc.,Falcon TM ,目录号:352096和352070"。
  2. 1ml无针注射器(Restek Corporation,目录号:22766)
  3. 100×1ml具有(28个1/2)针头(BD,目录号:329420)的注射器
  4. 50x NOD-SCID或NSG小鼠
  5. ?5ml基质胶(Corning,目录号:354248)
  6. 洗涤介质(DMEM/F-12)(Thermo Fisher Scientific,Gibco TM ,目录号:11320-033)
  7. 0.4%台盼蓝溶液(Life Technologies,目录号:15250-061)
    注意:目前,"赛默飞世尔科技,Gibco TM <目录号:15250-061"
  8. 0.25%胰蛋白酶(Life Technologies,目录号:25200-056) 注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:25200-056"
  9. 青霉素 - 链霉素 - 两性霉素B(1%)(Life technologies,目录号:15240-062)
    注意: 目前,它是"抗生素 - 抗真菌剂(100x)(Thermo Fisher Scientific,Gibco TM >,目录号:15240-062)"。
  10. L-谷氨酰胺(2mM)(Invitrogen,目录号:25030081) 注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:25030081" />
  11. 非必需氨基酸(1x)(Thermo Fisher Scientific,Hyclone TM ,目录号:SH3023801)
  12. 丙酮酸钠(1mM)(Thermo Fisher Scientific,Hyclone TM,目录号:SH3023901)
  13. HEPES(1x)(Invitrogen,目录号:15630080) 注意:目前,"赛默飞世尔科技,Gibco TM ,目录号:15630080" />
  14. 来自猪肠粘膜的肝素钠盐(4μg/ml)(Sigma-Aldrich,目录号:H3149-100KU)
  15. 脂质(Sigma-Aldrich,目录号:L0288)
  16. 人表皮生长因子重组体(EGF)(20ng/ml)(Biomartcanada,目录号:RKP01133)
  17. 人碱性成纤维细胞生长因子重组体(bFGF)(10ng/ml)(Biomartcanada,目录号:RKP09038)
  18. N 2补充物(STEMCELL Technologies,目录号:07152)
  19. NeuroCult SM1神经元(STEMCELL Technologies,目录号:05711)
  20. 无血清细胞培养基(参见配方)

设备

  1. 37°C孵育器
  2. 光学显微镜(Leica Microsystems,型号:DM IL LED)
  3. 血球计(Hausser Scientific,目录号:1483)
  4. 吸移助剂(INTEGRA Biosciences AG,??目录号:155000)
  5. 移液管(BD Falcon,目录号:357530)
    注意:目前,"Corning Inc.,Falcon TM ,目录号:357530"。
  6. 鼠标耳冲(Kent Scientific Corporation,目录号:INS750075-5)

程序

注意:使用的细胞浓度范围将取决于您的部分中起始细胞的频率。使用宽范围浓度的初始LDA可以用于找到最佳的细胞剂量范围,理想地,其应该包括具有100%肿瘤形成的剂量,直到没有肿瘤形成的细胞剂量,以及在其间的剂量范围。为了使LDA在统计学上有效,必须首先测试它们的样品是否符合泊松单一命中模型,其假定培养物中的生物活性单元的数目根据泊松分布而变化。有关其他信息和计算,请参阅ELDA网站( http://bioinf.wehi.edu.au)/software/elda/)和随附的文件(Hu和Smyt,2009)。例如:如果启动频率约为(1:1,000 - 1:5,000),则表2中所示的范围将足以获得统计上显着的结果。

  1. 准备细胞注射器
    1. 在37℃下在T175烧瓶中培养细胞汇合
    2. 在室温下离心细胞(300-450×g)5分钟,然后除去上清液。
    3. 当细胞旋转时,在冰上解冻基质胶。
    4. 将沉淀重悬在?3ml胰蛋白酶中,并在37℃孵育必要的时间以获得单细胞。
    5. 从培养箱中取出细胞,并用?10ml的洗涤培养基重悬,以停止胰蛋白酶反应。
    6. 在室温下离心细胞(300-450×g)5分钟,然后除去上清液
    7. 完全重悬颗粒在足够的细胞培养基,以便你会 具有约3×10 6个细胞/ml的最终细胞浓度 使血细胞计数器上的细胞计数更容易。确保您的 ?细胞浓度不小于2×10 6个细胞/ml,以允许 细胞浓度。步骤A9中列出的细胞浓度
    8. 混合10微升的细胞混合物 用10μl台盼蓝并将混合物装载到血细胞计数器上进行计数 ?使用光学显微镜的活细胞数。一定要计数 单元格在所有四个坐标中除以2得到 溶液中细胞浓度(_×10 4/uml/ml)。请参阅 http://www.abcam.com/protocols/counting-细胞使用a-血细胞计数器 ?有关如何使用血细胞计数器的进一步说明
    9. 连续稀释细胞,使您具有以下最终细胞浓度
      1. 管1(50,000个细胞/注射):在360μl培养基中的600,000个细胞
      2. 管2(10,000个细胞/注射):在360μl培养基中的120,000个细胞
      3. 管3(1,000个细胞/注射):在360μl培养基中的12,000个细胞
      4. 管4(100个细胞/注射):在360μl培养基中的1200个细胞
      5. 管5(10个细胞/注射):在360μl培养基中有120个细胞
    10. 准备注射器(与针)通过删除柱塞和添加 必要的标签(即细胞浓度,治疗等)。
    11. 使用注射器(无针),吸出基质胶并沉积 小滴凝胶(?200μl)注射器的基部。
    12. 充分混合所需的细胞溶液,然后加入30μl到基质胶液滴
    13. 小心地重新插入柱塞,确保不将基质溶液从注射器中排出
    14. 保持加载的注射器在冰上,直到准备使用。

      表2.体内有限稀释测定的细胞浓度和注射
      细胞浓度
      (#cells/injection)
      注射次数
      (2次注射/小鼠)
      小鼠数量
      50,000
      20
      10
      10,000
      20
      10
      1,000
      20
      10
      100
      20
      10
      10
      20
      10
      总计:
      100
      50

    15. 重复此步骤,直到您有100个注射器(每个细胞浓度20个注射器),如表2所示。

  2. 注射小鼠
    1. 轻度镇静小鼠与异氟烷和耳朵陷阱他们识别每个 小鼠(参见Laboratory Animal Medicine,2015,更多信息 动物识别)。
    2. 用细胞皮下注射小鼠 右侧和左侧,每次注射使用一个新的注射器 (参见图2)。每只小鼠应该有两次注射,每侧各一次 ?(参见表2)。
    3. 将鼠回到笼子里恢复。


      图2.小鼠右侧和左侧的皮下注射部位


      图3. 体内 LDA示意图

  3. 在体内评分 LDA
    1. 在?4-8周后,确定哪些小鼠已经发展成肿瘤。
    2. 继续监测0-1个肿瘤的小鼠长达6个月,或长度 作为伦理允许,以确保没有晚期肿瘤形成。
    3. 使用ELDA:极限稀释分析在线软件 计算癌细胞起始频率和显着性 ( http://bioinf.wehi.edu.au/software/elda/) 。

笔记

  1. 重复LDA时,结果会略有不同,但是当比较治疗组时,LDA结果应每次传达相同的总体结果。例如;与对照治疗'B'相比,治疗'A'总是产生较低的,统计学显着的起始频率
  2. 在 i 和在将增加测定的精确性。

食谱

  1. 无血清细胞培养基
    DMEM/F-12(1:1比例),补充有:
    1. 青霉素 - 链霉素 - 两性霉素B(1%)5ml/500ml
    2. L-谷氨酰胺(2mM)5ml/500ml
    3. 非必需氨基酸(1x)5ml/500ml
    4. 丙酮酸钠(1mM)5ml/500ml
    5. HEPES(1x)5ml/500ml
    6. 来自猪肠粘膜的肝素钠盐(4μg/ml)
    7. 脂质1ml/500ml
    8. 人表皮生长因子重组体(EGF)(20ng/ml)
    9. 人碱性成纤维细胞生长因子重组体(bFGF)(10ng/ml)
    10. N 2补充物-5ml/500ml
    11. NeuroCult SM1神经元2ml/500ml

参考文献

  1. 使用血细胞计数器计数细胞: Abcam。
  2. Fazekas de St,G。(1982)。 有限稀释分析的评估。 J Immunol方法 49(2):R11-23。
  3. Hu,Y。和Smyth,G.K。(2009)。 ELDA:极限稀释分析,用于比较干细胞和其他测定中的耗尽和富集的群体。 a Immunol Methods 347(1-2):70-78。
  4. Kreso,A.,van Galen,P.,Pedley,NM,Lima-Fernandes,E.,Frelin,C.,Davis,T.,Cao,L.,Baiazitov,R.,Du,W.,Sydorenko,N 。,Moon,YC,Gibson,L.,Wang,Y.,Leung,C.,Iscove,NN,Arrowsmith,CH,Szentgyorgyi,E.,Gallinger,S.,Dick,JE和O'Brien, )。 自我更新作为人类结肠直肠癌的治疗靶点 Nat Med 20(1):29-36。
  5. Strijbosch,L.W.,Buurman,W.A.,Does,R.J.,Zinken,P.H.and Groenewegen,G。(1987)。 限制稀释测定。实验设计和统计分析。 J Immunol Methods 97(1):133-140。
  6. Talcott,M.,Akers,W.,and Marini,R。(2015)。实验技术。在:Anderson,L。和Otto,G。(eds)。 实验动物医学 Elsevier,1202-1204。
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引用:Agro, L. and O’Brien, C. A. (2015). In vitro and in vivo Limiting Dilution Assay for Colorectal Cancer. Bio-protocol 5(22): e1659. DOI: 10.21769/BioProtoc.1659.
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