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Melanoma Stem Cell Sphere Formation Assay
黑色素瘤干细胞球体形成测定   

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Abstract

Self-renewal is the ability of cells to replicate themselves at every cell cycle. Throughout self-renewal in normal tissue homeostasis, stem cell number is maintained constant throughout life. Cancer stem cells (CSCs) share this ability with normal tissue stem cells and the sphere formation assay (SFA) is the gold standard assay to assess stem cells (or cancer stem cells) self-renewal potential in vitro. When single cells are plated at low density in stem cell culture medium, only the cells endowed with self-renewal are able to grow in tridimensional clusters usually named spheres. In the recent years, SFA has been used also to test the effect of several drugs, chemical and natural compounds or microenviromental components on stem cells self-renewal capacity. Here we will illustrate a detailed protocol to assess self-renewal of human melanoma stem cells, growing as melanospheres.

Keywords: Melanoma(黑色素瘤), Cancer stem cells(癌症干细胞), Melanospheres(黑色素瘤细胞球), Self-renewal(自我更新), Stem cell medium(干细胞培养基)

Background

Cancer stem cells (CSCs) were first found in acute myeloma leukemia (Lapidot et al., 1994) and then were identified in many solid tumors including melanoma. CSCs are defined as cells strongly endowed with self-renewal and tumor initiating capacity, being able to regenerate the whole tumor heterogeneity in vivo. CSCs can be isolated from the tumor mass with different approaches based on phenotypic characteristic or biological properties, then their properties have to be tested in vitro (self-renewal) and in vivo (tumorigenic potential). Melanoma CSCs were isolated using a combination of cell surface markers, (Fang et al., 2005; Monzani et al., 2007; Schatton et al., 2008; Boiko et al., 2010; Boonyaratanakornkit et al., 2010) or through culture in specific stem cell media (Perego et al., 2010; Santini et al., 2012). To validate melanoma CSC self-renewal, and to study the effect of tumor microenvironmental factors on it (Tuccitto et al., 2016), we used the sphere formation assay (SFA) in vitro. Melanoma CSCs are plated at low density in stem cell culture medium and they grow in anchorage-independent, three-dimensional spherical structures, called melanospheres (tumorspheres, in general). Spheres forming efficiency is directly proportional to the number of melanoma CSCs present in the culture (one CSC corresponds to one melanosphere), thus giving a direct quantification of CSC amount in culture. This relatively simple method is useful to study the ability of any exogenous factors (growth factors, cytokines and chemokines, drugs) in perturbing CSC self-renewal (Tsuyada et al., 2012; Tuccitto et al., 2016). Here we provide detailed information about the SFA protocol we optimized in our laboratory for melanoma SFA.

Materials and Reagents

  1. Cell culture flask, area 150 cm2 (Corning, catalog number: 430823 )
  2. 15 ml Falcon tube (Greiner Bio One International, catalog number: 188261 )
  3. Micropipette P200 tips (Corning, catalog number: 4823 )
  4. 24-wells plates flat bottom (Corning, Costar®, catalog number: 3527 )
  5. 70 μm cell strainer (Corning, Falcon®, catalog number: 352350 )
  6. 15 ml polystyrene serological pipets (Corning, Falcon®, catalog number: 357551 )
  7. Melanospheres are obtained as described by Perego et al., starting from cell suspension obtained from melanoma surgical specimens or from previously established melanoma cell lines after culturing in stem cell medium (SCM) (Perego et al., 2010)
  8. RPMI
  9. 10% FBS
  10. Trypan blue solution, 0.4% (Sigma-Aldrich, catalog number: T8154 )
  11. SCM (see Recipes)
    1. DMEM:F-12, 1:1 mixture’ (Lonza, catalog number: BE12-719F )
    2. Epidermal growth factor (EGF) (PeproTech, catalog number: AF-100-15 )
    3. Basic fibroblast growth factor (bFGF) (PeproTech, catalog number: 100-18B )
    4. D-glucose (Sigma-Aldrich, catalog number: G7021 )
    5. Insulin (Sigma-Aldrich, catalog number: I6634 )
    6. Putrescine dihydrochloride (Sigma-Aldrich, catalog number: P5780 )
    7. Sodium selenite (Sigma-Aldrich, catalog number: S9133 )
    8. Progesterone (Sigma-Aldrich, catalog number: P6149 )
    9. Transferrin (Sigma-Aldrich, catalog number: T8158 )
    10. Sodium bicarbonate (NaHCO3) (Sigma-Aldrich, catalog number: S8761 )
    11. 1x phosphate buffered saline (PBS) (Lonza, catalog number: 17-516 )
    12. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A1933 )
    13. HEPES buffer (Lonza, catalog number: BE17-737 )
    14. L-glutamine (Lonza, catalog number: BE17-605E )
    15. Penicillin-streptomycin (Lonza, catalog number: 17-602E )

Equipment

  1. Automatic pipettor (PBI, catalog number: 857075 )
  2. Tissue culture incubator with CO2 input (Thermo Fisher Scientific, Thermo ScientificTM, model: FormaTM Series II 3110 )
  3. Centrifuge (Eppendorf, catalog number: 5810 )
  4. Hemocytometer (Marienfeld-Superior, Bürker, catalog number: 0640211 )
  5. Optical microscope (Carl Zeiss, model: Axiovert 25 )

Procedure

Notes:

  1. Work in tissue culture hood with sterile tools and equipment.
  2. This procedure was optimized for melanoma cells. The general principle could be applied to cancer stem cells for other tissues, with optimization.
  1. Prepare stem cell medium (SCM) and store it at 4 °C in the dark. SCM should be used within 3-4 weeks from preparation. It is also possible to prepare a concentrated solution (10x) with all the supplements (DMEM:F-12, 1:1 mixture supplemented with components listed above in the Materials and Reagents section from 6b to 6o). The concentrated medium can be stored at 20 °C up to 3 months. Growth factors (b-FGF and EGF) have to be added fresh directly to the medium.
    Note: If you are planning to set up a sphere formation assay (SFA) with cells not cultured as melanospheres (adherent melanoma cells are grown in RPMI + 10% FBS, fresh melanoma tissue suspension is obtained from the processing of melanoma post-surgery specimens), start from step 10 of this procedure. In case of cells cultured with serum, be sure all the serum is removed (wash your cells extensively) before plating in SCM.
  2. Collect melanospheres from the culture flask in a 15 ml Falcon tube.
  3. Centrifuge for 10 min at 30 x g at room temperature (RT).
  4. Discard the supernatant.
  5. Dissociate the pellet by pipetting up and down using a micropipette P200 tips for about 80-100 times in 100-200 μl of SCM.
    Note: To dissociate the pellet, place the tip at the bottom of the tubes and pipette up and down fast enough to suspend the pellet and the cells without making bubble (too gentle pipetting will result in not complete sphere dissociation). In our hands, mechanical dissociation was the best method in terms of cell viability and results reproducibility to obtain single cell suspension.
  6. Suspend the dissociated melanospheres in 2 ml SCM.
  7. Centrifuge for 10 min at 150 x g at room temperature (RT) and discard the supernatant.
  8. Dissociate the pellet by pipetting up and down using a micropipette P200 tips for about 80-100 times.
  9. Suspend the dissociated melanospheres in SCM and count.
    Note: Make sure to obtain a single cell suspension. If clusters are still present, repeat steps 6-9. Eventually the cell suspension could be further filtered through a 70 μm cell strainer.
  10. Take the required number of live cells (determined after counting with a hemocytometer in presence of trypan blue solution 0.4% to exclude dead cells) and adjust to a final concentration of 100 cells/100 µl in SCM.
  11. Pipette 400 µl/well of SCM in each 24-well plate for a total of 6 wells for each cell line or treatment.
    Note: In our hands, SFA was largely used to test cytokines and chemokines effect on melanoma stem cells self-renewal (Tuccitto et al., 2016). We think that melanoma SFA could be optimized also to test the effects of drugs (targeting stem cells markers or active signaling pathways) or growth factors on melanoma stem cell self-renewal and or viability.
  12. Seed 100 µl of the cells in SCM (100 cells/well) for each well, prepare at least 4-6 replicates for each condition. The seeded cells should appear as singlets (see Figure 1A).


    Figure 1. Melanoma sphere formation. A. Single cell melanospheres plated in SCM; B. Doublets appearing when cells start dividing; C. Triplets formed during cell proliferation; D. Fully formed melanospheres. Total magnification 200x (A-D), scale bar = 20 µm.

  13. Place the plate in an incubator set at 37 °C with 5% CO2.
  14. 24-72 h after seeding, the cells doublets and triplets will appear in wells and could be counted under an optical microscope (see Figures 1B and 1C).
    Note: If the media turns yellow, add fresh media to the well without dissociating or touching the ongoing forming spheres.
  15. Melanospheres will be completely formed in each well 5-8 days after seeding and can be counted again under an optical microscope with 10x objective (total magnification 100x) analyzing the entire well. If spheres are bigger enough to be clearly distinguished from monolayer aggregates and single cells, also lower magnification should be used (20-50x total magnification). For better results, count melanospheres when they are clearly visible and do not let them over-proliferate. If this happen, melanospheres could attach to the plate and reliable counting will be difficult.
    Note: When melanospheres are formed (see Figure 1D) it is difficult to distinguish the exact number of cells due to their compact 3D structure. Occasionally, some monolayer aggregates of about 10-30 cells could be observed, but those aggregates shouldn’t be accounted as melanospheres (Figure 2). Generally, spheres could be counted around day 5 up to day 10 from SFA seeding, optimal counting time should be determined for each cell line or sample and then maintained constant across different experiments. To simplify the count of the spheres it could be useful drawing some line with a marker to subdivide the well into quarters.


    Figure 2. Non-melanosphere clusters. Example of non-spherical cluster that could be found in SFA. Those clusters could not be counted as spheres. Total magnification 200x, scale bar = 20 µm.

Data analysis

Sphere efficiency can be reported as percentage (%) of spheres formed dividing by the original number of cells seeded or number of spheres counted considering the total number of single cells seeded. We usually presented the results as mean ± SD (or SEM) of biological replicates. Each cell line was tested multiple times to be sure that the sphere forming efficiency was maintained after extensive cell culture.

Notes

  1. Self-renewal efficiency it is cell-type or cell-line dependent, depending on the melanospheres origin. In our hands, melanospheres showed between 30% and 15% of sphere forming efficiency (Perego et al., 2010; Tuccitto et al., 2016).
  2. Self-renewal efficiency is generally higher for cells maintained in culture as melanospheres than for cells previously cultured in regular melanoma culture medium with serum (adherent melanoma cultures). Melanospheres cultures are already enriched in CSC compared to adherent culture, so SFA from adherent cell lines would result in lower sphere forming efficiency, in general.

Recipes

  1. Stem cell medium (SCM) (500 ml)
    DMEM:F-12 1:1 mixture
    10 µg/ml EGF
    5 µg/ml bFGF
    600 µg/ml D-glucose
    5 µg/ml insulin
    1 µg/ml putrescine dihydrochloride
    0.01 µg/ml sodium selenite
    0.01 µg/ml progesterone
    20 µg/ml transferrin
    0.11 µg/ml NaHCO3
    4 mg/ml BSA
    119.2 µg/ml HEPES buffer
    292 µg/ml L-glutamine
    200 UI/ml penicillin-streptomycin
    Notes:
    1. 10x BSA solution in 1x PBS can be stored at -20 °C.
    2. If stock solution are made, make sure to respect the final volume of 500 ml, otherwise some components will be diluted and the media should not work.

Acknowledgments

This protocol was used in Perego et al., 2010 and Tuccitto et al., 2016. These works were supported by the Associazione Italiana Ricerca sul Cancro (CC IG-10615).

References

  1. Boiko, A. D., Razorenova, O. V., van de Rijn, M., Swetter, S. M., Johnson, D. L., Ly, D. P., Butler, P. D., Yang, G. P., Joshua, B., Kaplan, M. J., Longaker, M. T. and Weissman, I. L. (2010). Human melanoma-initiating cells express neural crest nerve growth factor receptor CD271. Nature 466(7302): 133-137.
  2. Boonyaratanakornkit, J. B., Yue, L., Strachan, L. R., Scalapino, K. J., LeBoit, P. E., Lu, Y., Leong, S. P., Smith, J. E. and Ghadially, R. (2010). Selection of tumorigenic melanoma cells using ALDH. J Invest Dermatol 130(12): 2799-2808.
  3. Fang, D., Nguyen, T. K., Leishear, K., Finko, R., Kulp, A. N., Hotz, S., Van Belle, P. A., Xu, X., Elder, D. E. and Herlyn, M. (2005). A tumorigenic subpopulation with stem cell properties in melanomas. Cancer Res 65(20): 9328-9337.
  4. Lapidot, T., Sirard, C., Vormoor, J., Murdoch, B., Hoang, T., Caceres-Cortes, J., Minden, M., Paterson, B., Caligiuri, M. A. and Dick, J. E. (1994). A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature 367(6464): 645-648.
  5. Monzani, E., Facchetti, F., Galmozzi, E., Corsini, E., Benetti, A., Cavazzin, C., Gritti, A., Piccinini, A., Porro, D., Santinami, M., Invernici, G., Parati, E., Alessandri, G. and La Porta, C. A. (2007). Melanoma contains CD133 and ABCG2 positive cells with enhanced tumourigenic potential. Eur J Cancer 43(5): 935-946.
  6. Perego, M., Tortoreto, M., Tragni, G., Mariani, L., Deho, P., Carbone, A., Santinami, M., Patuzzo, R., Mina, P. D., Villa, A., Pratesi, G., Cossa, G., Perego, P., Daidone, M. G., Alison, M. R., Parmiani, G., Rivoltini, L. and Castelli, C. (2010). Heterogeneous phenotype of human melanoma cells with in vitro and in vivo features of tumor-initiating cells. J Invest Dermatol 130(7): 1877-1886.
  7. Santini, R., Vinci, M. C., Pandolfi, S., Penachioni, J. Y., Montagnani, V., Olivito, B., Gattai, R., Pimpinelli, N., Gerlini, G., Borgognoni, L. and Stecca, B. (2012). Hedgehog-GLI signaling drives self-renewal and tumorigenicity of human melanoma-initiating cells. Stem Cells 30(9): 1808-1818.
  8. Schatton, T., Murphy, G. F., Frank, N. Y., Yamaura, K., Waaga-Gasser, A. M., Gasser, M., Zhan, Q., Jordan, S., Duncan, L. M., Weishaupt, C., Fuhlbrigge, R. C., Kupper, T. S., Sayegh, M. H. and Frank, M. H. (2008). Identification of cells initiating human melanomas. Nature 451(7176): 345-349.
  9. Tsuyada, A., Chow, A., Wu, J., Somlo, G., Chu, P., Loera, S., Luu, T., Li, A. X., Wu, X., Ye, W., Chen, S., Zhou, W., Yu, Y., Wang, Y. Z., Ren, X., Li, H., Scherle, P., Kuroki, Y. and Wang, S. E. (2012). CCL2 mediates cross-talk between cancer cells and stromal fibroblasts that regulates breast cancer stem cells. Cancer Res 72(11): 2768-2779.
  10. Tuccitto, A., Tazzari, M., Beretta, V., Rini, F., Miranda, C., Greco, A., Santinami, M., Patuzzo, R., Vergani, B., Villa, A., Manenti, G., Cleris, L., Giardiello, D., Alison, M., Rivoltini, L., Castelli, C. and Perego, M. (2016). Immunomodulatory factors control the fate of melanoma tumor initiating cells. Stem Cells 34(10): 2449-2460.

简介

自我更新是细胞在每个细胞周期复制自身的能力。在正常组织体内平衡的自我更新过程中,干细胞数量在整个生命中保持不变。癌症干细胞(CSCs)与正常组织干细胞共享这种能力,球形成测定(SFA)是评估干细胞(或癌症干细胞)体外自我更新潜力的金标准测定方法。 。当单细胞在干细胞培养基中以低密度铺板时,仅具有自我更新的细胞能够在通常称为球体的三维簇中生长。近年来,SFA也用于测试几种药物,化学和天然化合物或微环境成分对干细胞自我更新能力的影响。在这里,我们将说明一个详细的方案来评估人类黑色素瘤干细胞的自我更新,作为黑色球体生长。

癌症干细胞(CSCs)首先在急性骨髓瘤白血病(Lapidot et al。,1994)中发现,然后在许多实体瘤中鉴定出包括黑素瘤。 CSCs被定义为具有自我更新和肿瘤起始能力的细胞,能够在体内再生整个肿瘤异质性。可以使用基于表型特征或生物学特性的不同方法从肿瘤块中分离CSCs,然后在体外(自我更新)和体内 (致瘤潜力)。使用细胞表面标志物的组合分离黑素瘤CSCs(Fang等人,2005; Monzani等人,2007; Schatton等人, ,2008; Boiko等人,2010; Boonyaratanakornkit等人,2010)或通过特定干细胞培养基中的培养(Perego等人, ,2010; Santini 等人,2012)。为了验证黑素瘤CSC自我更新,并研究肿瘤微环境因子对其的影响(Tuccitto等,2016),我们在体外使用球形成分析(SFA)。黑素瘤CSC以低密度电镀在干细胞培养基中,并且它们在与锚定无关的三维球形结构中生长,称为黑色球体(通常为肿瘤球体)。球形成效率与存在于培养物中的黑素瘤CSC的数量成正比(一个CSC对应于一个黑色素层),从而直接量化培养物中的CSC量。这种相对简单的方法可用于研究任何外源因子(生长因子,细胞因子和趋化因子,药物)扰乱CSC自我更新的能力(Tsuyada等,2012; Tuccitto et et al。 ,,2016)。在这里,我们提供我们在我们实验室中优化的黑色素瘤SFA的SFA方案的详细信息。

关键字:黑色素瘤, 癌症干细胞, 黑色素瘤细胞球, 自我更新, 干细胞培养基

材料和试剂

  1. 细胞培养瓶,面积150cm 2(Corning,目录号:430823)
  2. 15 ml Falcon管(Greiner Bio One International,目录号:188261)
  3. Micropipette P200提示(Corning,目录号:4823)
  4. 24孔板平底(Corning,Costar ®,目录号:3527)
  5. 70μm细胞过滤器(Corning,Falcon ®,目录号:352350)
  6. 15ml聚苯乙烯血清移液管(Corning,Falcon ®,目录号:357551)
  7. 如从Perego等人所述获得黑素球,从获自黑素瘤手术标本的细胞悬浮液或从干细胞培养基(SCM)培养后的先前建立的黑素瘤细胞系开始(Perego等人,,2010)
  8. RPMI
  9. 10%FBS
  10. 台盼蓝溶液,0.4%(Sigma-Aldrich,目录号:T8154)
  11. SCM(见配方)
    1. DMEM:F-12,1:1混合物(Lonza,目录号:BE12-719F)
    2. 表皮生长因子(EGF)(PeproTech,目录号:AF-100-15)
    3. 碱性成纤维细胞生长因子(bFGF)(PeproTech,目录号:100-18B)
    4. D-葡萄糖(Sigma-Aldrich,目录号:G7021)
    5. 胰岛素(Sigma-Aldrich,目录号:I6634)
    6. 二盐酸腐胺(Sigma-Aldrich,目录号:P5780)
    7. 亚硒酸钠(Sigma-Aldrich,目录号:S9133)
    8. 孕激素(Sigma-Aldrich,目录号:P6149)
    9. 转铁蛋白(Sigma-Aldrich,目录号:T8158)
    10. 碳酸氢钠(NaHCO 3)(Sigma-Aldrich,目录号:S8761)
    11. 1x磷酸缓冲盐水(PBS)(Lonza,目录号:17-516)
    12. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A1933)
    13. HEPES缓冲区(Lonza,目录号:BE17-737)
    14. L-谷氨酰胺(Lonza,目录号:BE17-605E)
    15. 青霉素 - 链霉素(Lonza,目录号:17-602E)

设备

  1. 自动移液器(PBI,目录号:857075)
  2. 具有CO 2输入的组织培养培养箱(Thermo Fisher Scientific,Thermo Scientific TM,型号:Forma TM系列II 3110)
  3. 离心机(Eppendorf,目录号:5810)
  4. 血细胞计数器(Marienfeld-Superior,Bürker,目录号:0640211)
  5. 光学显微镜(Carl Zeiss,型号:Axiovert 25)

程序

注意:

  1. 用无菌工具和设备在组织培养罩中工作。
  2. 该过程针对黑素瘤细胞进行了优化。一般原则可以适用于其他组织的癌症干细胞,优化。
  1. 准备干细胞培养基(SCM)并将其在黑暗中保存在4°C。 SCM应在3-4周内使用。还可以制备所有补充剂(DMEM:F-12,1:1混合物,补充上述在材料和试剂部分中的组分从6b至6o)的浓缩溶液(10x)。浓缩的培养基可以在20℃下储存3个月。生长因子(b-FGF和EGF)必须直接添加到培养基中 注意:如果您打算用不作为黑色球体培养的细胞建立球形形成测定(SFA)(粘附的黑素瘤细胞在RPMI + 10%FBS中生长,则从黑素瘤后处理获得新鲜的黑素瘤组织悬浮液手术标本),从本程序的第10步开始。在用血清培养的细胞的情况下,在SCM中电镀前,确保除去所有血清(大量洗涤细胞)。
  2. 从15 ml Falcon管中的培养瓶中收集黑色球。
  3. 在室温(RT)下以30×g离心10分钟。
  4. 丢弃上清液。
  5. 通过使用微量移液管P200尖端在100-200μlSC​​M中上下移动约80-100次来分离沉淀物。
    注意:为了分离沉淀物,将尖端放置在管的底部,并将吸管快速上下移动,以便悬浮颗粒和细胞而不会发生气泡(太平缓的移液将导致未完全的球体解离)。在我们手中,机械解离是获得单细胞悬浮液的细胞活力和结果再现性方面最好的方法。
  6. 将解离的黑色球悬浮于2ml SCM中
  7. 在室温(RT)下以150×g离心10分钟,弃去上清液。
  8. 通过使用微量移液管P200尖端上下移动约80-100次来分离沉淀。
  9. 在SCM中悬浮解离的黑色球体并计数。
    注意:确保获得单细胞悬浮液。如果簇仍然存在,请重复步骤6-9。最终细胞悬浮液可以通过70μm的细胞过滤器进一步过滤。
  10. 取所需数量的活细胞(使用血细胞计数器计数,在台盼蓝溶液0.4%存在以排除死细胞后测定),并在SCM中调整至100细胞/100μl的终浓度。
  11. 在每个24孔板中移取400μl/孔的SCM,每个细胞系或治疗总共6个孔。
    注意:在我们手中,SFA主要用于测试细胞因子和趋化因子对黑素瘤干细胞自我更新的影响(Tuccitto et al。,2016)。我们认为可以优化黑素瘤SFA以测试药物(靶向干细胞标志物或活性信号通路)或生长因子对黑素瘤干细胞自我更新和/或活力的影响。
  12. 在每个孔中种子100微升的SCM细胞(100个细胞/孔),每个条件至少准备4-6个重复。种子细胞应显示为单数(见图1A)。


    图1.黑色素瘤球形成。 A.单细胞黑色球体接种在SCM中;当细胞开始分裂时出现双峰; C.细胞增殖过程中形成的三倍体; D.完全形成的黑色球。总倍率200倍(A-D),比例尺= 20微米
  13. 将板置于37℃的培养箱中,5%CO 2
  14. 接种后24-72小时,细胞双峰,三联体将出现在孔中,并可在光学显微镜下计数(参见图1B和1C)。
    注意:如果介质变黄,请将新鲜介质添加到孔中,而不会分离或接触正在进行的成型球体。
  15. 播种后5-8天,每孔中将会完全形成黑色球体,并且可以在光学显微镜下再次计数10倍物镜(总放大100倍),分析整个孔。如果球体足够大以与单层聚集体和单个细胞清楚区分,还应使用较低的放大倍率(总放大倍数为20-50倍)。为了获得更好的效果,当它们清晰可见并且不让它们过度增殖时,计数黑色球体。如果发生这种情况,黑色球体可能附着在板上,可靠的计数将是困难的。
    注意:当黑斑形成时(见图1D),由于其紧凑的3D结构,难以区分细胞的确切数量。偶尔,可以观察到约10-30个细胞的一些单层聚集体,但是这些聚集体不应被视为黑色球体(图2)。一般来说,球蛋白可以在第5天到第10天从SFA播种计数,应该为每个细胞系或样品确定最佳计数时间,然后在不同的实验中保持恒定。为了简化球体的计数,可以用一个标记来绘制一些线,将井细分为四分之一。


    图2.非黑洞群集可以在SFA中找到的非球形簇的示例。这些群集不能算作球体。总倍率200倍,比例尺= 20微米。

数据分析

球形效率可以报告为除以原始接种细胞数的球体百分数(%),或考虑到单株细胞总数计算的球数。我们通常将结果呈现为生物重复的平均值±SD(或SEM)。每个细胞系被多次测试以确保在广泛细胞培养后保持球形成效率。

笔记

  1. 自我更新效率是细胞型或细胞依赖性,取决于黑色球起源。在我们手中,黑色球显示球形成效率的30%至15%(Perego等人,2010; Tuccitto等人,2016)。
  2. 对于维持在黑色球体的培养物中的细胞,与先前在具有血清(粘附黑素瘤培养物)的常规黑素瘤培养基中培养的细胞相比,自我更新效率通常更高。与粘附培养相比,黑色球培养物已经富集CSC,因此粘附细胞系的SFA通常会导致较低的球体形成效率。

食谱

  1. 干细胞培养基(SCM)(500毫升)
    DMEM:F-12 1:1混合物 10μg/ml EGF
    5μg/ml bFGF
    600μg/ml D-葡萄糖
    5μg/ml胰岛素 1μg/ml腐胺二盐酸盐 0.01微克/毫升亚硒酸钠
    0.01μg/ml孕酮
    20μg/ml转铁蛋白
    0.11μg/ml NaHCO 3
    4毫克/毫升BSA
    119.2μg/ml HEPES缓冲液
    292μg/ml L-谷氨酰胺
    200 UI/ml青霉素 - 链霉素
    注意:
    1. 1×PBS中的10x BSA溶液可以储存在-20°C。
    2. 如果采用储备溶液,请确保最终体积达到500毫升,否则一些组分将被稀释,介质不能起作用。

致谢

该协议在2010年的Perego等人,2010年和Tuccitto等人,2016中使用。这些作品得到了意大利Ricerca sul Cancro(CC IG-10615)的支持, 。

参考文献

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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Tuccitto, A., Beretta, V., Rini, F., Castelli, C. and Perego, M. (2017). Melanoma Stem Cell Sphere Formation Assay. Bio-protocol 7(8): e2233. DOI: 10.21769/BioProtoc.2233.
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