生命科学

采用细胞示踪染料进行细胞分裂监测的优化染色和增殖模型方法

荧光细胞示踪染料,通过与流式细胞仪和图像细胞仪的结合,已成为在体内和体外对不同细胞类型的相互作用和命运研究的一种有力工具。1-5 尽管已有上千篇的文献在引用这样的染料,一些最为通常涉及的细胞示踪引用包括对于过程的监测:

  1. 干细胞和始祖细胞的休眠、扩增及分化6-8
  2. 抗原驱动的膜转移9和前体细胞的扩增3,4,10-18以及
  3. 免疫调节和调节因子细胞功能1,18-21

目前市场上细胞示踪染料在其化学性质和荧光性质方面差别很大,但绝大多数细胞示踪染料可根据它们的细胞标记机制分为两类。以PKH26为代表的“膜染料”是部分稳定但与细胞膜发生非共价结合的高度亲脂性的染料。1,2,11以CFSE为代表的“蛋白染料”是可与细胞蛋白形成稳定共价键的氨基活性染料4,16,18。这两类染料各自都有其优点和不足。对它们进行成功的利用,特别是需要使用多种染料来对不同类型细胞进行示踪的多色研究,关键在于了解如何可以促进每种类型染料优化使用的关键问题2-4,16,18,24

这里所包含的操作方法对使用细胞示踪染料时三种可引起不良或变异结果的主要因素进行了强调:

  1. 无法获得明亮、均一且可重复的标记
  2. 这是进行任何细胞示踪研究关键的第一步,特别是相对于使用蛋白染料或平衡结合试剂如抗体时,在使用膜染料时需要特别注意所产生的变异。
  3. 荧光染料复合效果不佳,或无法包含重要的补偿对照。
  4. 示踪染料荧光通常是抗体荧光亮度的100至1000倍。因此需要确保示踪染料的存在不会影响对其他所使用探针的检测。
  5. 通过峰模型软件无法获得较好的拟合性。
  6. 这些软件可通过前体的频率或其他指标来对不同群体或刺激对增殖反应进行量化的比较。获取较好的拟合性需要排除可影响染料稀释谱的死细胞或将死细胞进行排除,并将观察到的染料稀释谱的特征与模型的假设进行匹配。

这里所给出的例子展示了在使用膜或蛋白染料来对细胞增殖进行监测时,这些变异将会如何影响结果。

 

材料

     

参考文献

  1. Poon, R.Y., Ohlsson-Wilhelm, B.M., Bagwell, C.B., & Muirhead, K.A. Use of PKH Membrane Intercalating Dyes to Monitor Cell Trafficking and Function. In: Living Color: Flow Cytometry and Cell Sorting Protocols., Diamond, R.A. & DeMaggio, S., eds., Springer-Verlag, New York, NY, 302-352 (2000).
  2. Wallace, P.K. & Muirhead, K.A. Cell Tracking 2007: A Proliferation of Probes and Applications. Immunol. Invest. 36, 527-562 (2007).
  3. Hawkins, E.D., Hommel, M., Turner, M.L., Battye, F.L., Markham, J.F., & Hodgkin, P.D. Measuring lymphocyte proliferation, survival and differentiation using CFSE time-series data. Nat. Protoc. 2, 2057-2067 (2007).
  4. Quah, B.J., Warren, H.S., & Parish, C.R. Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester. Nat. Protoc. 2, 2049-2056 (2007).
  5. Bolton, D.L., Minang, J.T., Trivett, M.T., Song, K., Tuscher, J.J., Li, Y., Piatak, Jr., M., O'Connor, D., Lifson, J.D., Roederer, M., & Ohlen, C. Trafficking, Persistence, and Activation State of Adoptively Transferred Allogeneic and Autologous Simian Immunodeficiency Virus-Specific CD8+ T Cell Clones during Acute and Chronic Infection of Rhesus MacaquesJ. Immunol. 184, 303-314 (2010).
  6. Juopperi, T.A. & Sharkis, S.J. Isolation of Quiescent Murine Hematopoietic Stem Cells by Homing PropertiesMeth. Mol. Biol. 430, 21-30 (2008).
  7. Kusumbe, A.P. & Bapat, S.A. Cancer stem cells and aneuploid populations within developing tumors are the major determinants of tumor dormancyCancer Res. 69, 9245-53 (2009).
  8. Pece, S., Tosonim, D., Confalonieri, S., Mazzarol, G., Vecchi, M., Ronzoni, S., Bernard, L., Viale, G., Pelicci, P.G., & Di Fiore, P.P. Biological and Molecular Heterogeneity of Breast Cancers Correlates with Their Cancer Stem Cell ContentCell. 140, 62-73 (2010).
  9. Gertner-Dardenne, J., Poupot, M., Gray, B.D., & Fournié, J.-J. Lipophilic fluorochrome trackers of membrane transfers between immune cellsImmunol. Invest. 36, 665-685 (2007).
  10. Bercovici, N., Givan, A.L., Waugh, M.G., Fisher, J.L., Vernel-Pauillac, F., Ernstoff, M.S., Abastado, J.P., & Wallace, P.K. Multiparameter precursor analysis of T-cell responses to antigenJ. Immunol. Methods.276, 5-17 (2003).
  11. Givan, A.L., Fisher, J.L., Waugh, M.G., Bercovici, N., & Wallace, P.K. Use of cell-tracking dyes to determine proliferation precursor frequencies of antigen-specific T cellsMethods Mol. Biol. 263, 109-24 (2004).
  12. Schwaab, T., Tretter, C.P., Gibson, J.J., Cole, B.F., Schned, A.R., Harris, R., Fisher, J.L., Crosby, N., Stempkowski, L.M., Heaney, J.A., & Ernstoff, M.S. Tumor-related immunity in prostate cancer patients treated with human recombinant granulocyte monocyte-colony stimulating factor (GM-CSF)Prostate. 66(6), 667-674 (2006).
  13. Bantly, A.D., Gray, B.D., Breslin, E., Weinstein, E.G., Muirhead, K.A., Ohlsson-Wilhelm, B.M., & Moore, J.S. CellVue Claret, a New Far-Red Dye, Facilitates Polychromatic Assessment of Immune Cell Proliferation. Immunol. Invest. 36, 581-605 (2007).
  14. Givan, A.L. A flow cytometric assay for quantitation of rare antigen-specific T-cells: using cell-tracking dyes to calculate precursor frequencies for proliferation. Immunol. Invest. 36, 563-580 (2007).
  15. Tario, J.D., Jr., Gray, B.D., Wallace, S.S., Muirhead, K.A., Ohlsson-Wilhelm, B.M., & Wallace, P.K. Novel lipophilic tracking dyes for monitoring cell proliferationImmunol Invest. 36, 861-885 (2007).
  16. Wallace, P.K., Tario, Jr. J.D., Fisher, J.L., Wallace, S.S., Ernstoff, M.S., & Muirhead, K.A. Tracking Antigen-Driven Responses by Flow Cytometry: Monitoring Proliferation by Dye Dilution. Cytometry. 73A, 1019-1034 (2008).
  17. Barth, R.J., Fisher, D.A., Wallace, P.K. Channon, J.Y., Noelle, R.L., Gui, J., & Ernstoff, M.S. A Randomized Trial of Ex vivo CD40L Activation of a Dendritic Cell Vaccine in Colorectal Cancer Patients: Tumor-Specific Immune Responses Are Associated with Improved SurvivalClin. Cancer Res. 16, 5548-5556 (2010).
  18. Tario, J.D., Jr., Muirhead, K.A., Pan, D., Munson, M., & Wallace, P.K. Tracking Immune Cell Proliferation and Cytotoxic Potential Using Flow CytometryMeth. Mol. Biol. 699, 119-164 (2011).
  19. Fuse, S. & Underwood, E. Simultaneous Analysis of In Vivo CD8+ T Cell Cytotoxicity Against Multiple Epitopes using Multicolor Flow CytometryImmunol. Invest. 36, 829-845 (2007).
  20. Schütz, C., Fleck, M., Mackensen, A., Zoso, A., Halbritter, D., Schneck, J.P., & Oelke, M. Killer artificial antigen-presenting cells: a novel strategy to delete specific T cellsBlood. 111, 3546-3552 (2008).
  21. Zaritskaya, L., Shurin, M.R., Sayers, T.J., & Malyguine, A.M. New flow cytometric assays for monitoring cell-mediated cytotoxicityExpert Rev. Vaccines. 9, 601-616 (2010).
  22. Roederer, M. Interpretation of cellular proliferation data: Avoid the panglossianCytometry79A, 95-101 (2011).
  23. Personal communication, E. Breslin (Zynaxis Cell Science, Inc.).
  24. Quah, B.J.C. & Parish, C.R. The Use of Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) to Monitor Lymphocyte ProliferationJ. Vis. Exp. (44), e2259, doi:10.3791/2259 (2010).
  25. Sigma product bulletin for PKH26.
  26. Houlihan, D.D. & Newsome, P.N. Critical Review of Clinical Trials of Bone Marrow Stem Cells in Liver DiseaseGastroenterology. 135, 438-450 (2008).
  27. Brusko, T.M., Hulme, M.A., Myhr, C.B., Haller, M.J., & Atkinson, M.A. Assessing the In Vitro Suppressive Capacity of Regulatory T CellsImmunol. Invest. 36, 607-628 (2007).