Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:

  1. stem and progenitor cell quiescence, proliferation and/or differentiation6-8
  2. antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and
  3. immune regulatory and effector cell function1,18-21.

Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes," typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes," typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.

The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:

  1. Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.
  2. Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.
  3. Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.

Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Materials
Loading

References

1.
Poon RYM, Ohlsson-Wilhelm BM, Bagwell CB, Muirhead KA. 2000. Use of PKH Membrane Intercalating Dyes to Monitor Cell Trafficking and Function.302-352. http://dx.doi.org/10.1007/978-3-642-57049-0_26
2.
Wallace PK, Muirhead KA. 2007. Cell Tracking 2007: A Proliferation of Probes and Applications. Immunological Investigations. 36(5-6):527-561. http://dx.doi.org/10.1080/08820130701812584
3.
Hawkins ED, Hommel M, Turner ML, Battye FL, Markham JF, Hodgkin PD. 2007. Measuring lymphocyte proliferation, survival and differentiation using CFSE time-series data. Nat Protoc. 2(9):2057-2067. http://dx.doi.org/10.1038/nprot.2007.297
4.
Quah BJC, Warren HS, Parish CR. 2007. Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester. Nat Protoc. 2(9):2049-2056. http://dx.doi.org/10.1038/nprot.2007.296
5.
Bolton DL, Minang JT, Trivett MT, Song K, Tuscher JJ, Li Y, Piatak M, O?Connor D, Lifson JD, Roederer M, et al. 2010. 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 Macaques. J.I.. 184(1):303-314. http://dx.doi.org/10.4049/jimmunol.0902413
6.
Juopperi TA, Sharkis SJ. 2008. Isolation of Quiescent Murine Hematopoietic Stem Cells by Homing Properties.21-30. http://dx.doi.org/10.1007/978-1-59745-182-6_2
7.
Kusumbe AP, Bapat SA. 2009. Cancer Stem Cells and Aneuploid Populations within Developing Tumors Are the Major Determinants of Tumor Dormancy. Cancer Research. 69(24):9245-9253. http://dx.doi.org/10.1158/0008-5472.can-09-2802
8.
Pece S, Tosoni D, Confalonieri S, Mazzarol G, Vecchi M, Ronzoni S, Bernard L, Viale G, Pelicci PG, Di Fiore PP. 2010. Biological and Molecular Heterogeneity of Breast Cancers Correlates with Their Cancer Stem Cell Content. Cell. 140(1):62-73. http://dx.doi.org/10.1016/j.cell.2009.12.007
9.
Gertner-Dardenne J, Poupot M, Gray B, Fournié J. 2007. Lipophilic Fluorochrome Trackers of Membrane Transfers between Immune Cells. LIMM. 36(5):665-685. http://dx.doi.org/10.1080/08820130701674646
10.
Bercovici N, Givan AL, Waugh MG, Fisher JL, Vernel-Pauillac F, Ernstoff MS, Abastado J, Wallace PK. 2003. Multiparameter precursor analysis of T-cell responses to antigen. Journal of Immunological Methods. 276(1-2):5-17. http://dx.doi.org/10.1016/s0022-1759(03)00059-0
11.
Givan AL, Fisher JL, Waugh MG, Bercovici N, Wallace PK. Use of Cell-Tracking Dyes to Determine Proliferation Precursor Frequencies of Antigen-Specific T Cells.109-124. http://dx.doi.org/10.1385/1-59259-773-4:109
12.
Schwaab T, Tretter CP, Gibson JJ, Cole BF, Schned AR, Harris R, Fisher JL, Crosby N, Stempkowski LM, Heaney JA, et al. 2006. Tumor-related immunity in prostate cancer patients treated with human recombinant granulocyte monocyte-colony stimulating factor (GM-CSF). Prostate. 66(6):667-674. http://dx.doi.org/10.1002/pros.20266
13.
Bantly AD, Gray BD, Breslin E, Weinstein EG, Muirhead KA, Ohlsson-Wilhelm BM, Moore JS. 2007. CellVue® Claret, a New Far-Red Dye, Facilitates Polychromatic Assessment of Immune Cell Proliferation. Immunological Investigations. 36(5-6):581-605. http://dx.doi.org/10.1080/08820130701712461
14.
Givan AL. 2007. A Flow Cytometric Assay for Quantitation of Rare Antigen-Specific T Cells: Using Cell-Tracking Dyes to Calculate Precursor Frequencies for Proliferation. Immunological Investigations. 36(5-6):563-580. http://dx.doi.org/10.1080/08820130701683803
15.
Tario JD, Gray BD, Wallace SS, Muirhead KA, Ohlsson-Wilhelm BM, Wallace PK. 2007. Novel Lipophilic Tracking Dyes for Monitoring Cell Proliferation. Immunological Investigations. 36(5-6):861-885. http://dx.doi.org/10.1080/08820130701712933
16.
Wallace PK, Tario JD, Fisher JL, Wallace SS, Ernstoff MS, Muirhead KA. 2008. Tracking antigen-driven responses by flow cytometry: Monitoring proliferation by dye dilution. Cytometry. 73A(11):1019-1034. http://dx.doi.org/10.1002/cyto.a.20619
17.
Barth RJ, Fisher DA, Wallace PK, Channon JY, Noelle RJ, Gui J, Ernstoff MS. 2010. 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 Survival. Clinical Cancer Research. 16(22):5548-5556. http://dx.doi.org/10.1158/1078-0432.ccr-10-2138
18.
Tario JD, Muirhead KA, Pan D, Munson ME, Wallace PK. 2011. Tracking Immune Cell Proliferation and Cytotoxic Potential Using Flow Cytometry.119-164. http://dx.doi.org/10.1007/978-1-61737-950-5_7
19.
Fuse S, Usherwood E. 2007. Simultaneous Analysis ofIn VivoCD8+ T Cell Cytotoxicity Against Multiple Epitopes using Multicolor Flow Cytometry. Immunological Investigations. 36(5-6):829-845. http://dx.doi.org/10.1080/08820130701683753
20.
Schütz C, Fleck M, Mackensen A, Zoso A, Halbritter D, Schneck JP, Oelke M. 2008. Killer artificial antigen-presenting cells: a novel strategy to delete specific T cells. 111(7):3546-3552. http://dx.doi.org/10.1182/blood-2007-09-113522
21.
Zaritskaya L, Shurin MR, Sayers TJ, Malyguine AM. 2010. New flow cytometric assays for monitoring cell-mediated cytotoxicity. Expert Review of Vaccines. 9(6):601-616. http://dx.doi.org/10.1586/erv.10.49
22.
Roederer M. 2011. Interpretation of cellular proliferation data: Avoid the panglossian. Cytometry. 79A(2):95-101. http://dx.doi.org/10.1002/cyto.a.21010
23.
Breslin E. 2011. Personal Communication. Zynaxis Cell Science, Inc..
24.
Quah BJC, Parish CR. The Use of Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) to Monitor Lymphocyte Proliferation. JoVE.(44): http://dx.doi.org/10.3791/2259
25.
Product bulletin for PKH26. Sigma-Aldrich.
26.
Houlihan DD, Newsome PN. 2008. Critical Review of Clinical Trials of Bone Marrow Stem Cells in Liver Disease. Gastroenterology. 135(2):438-450. http://dx.doi.org/10.1053/j.gastro.2008.05.040
27.
Brusko TM, Hulme MA, Myhr CB, Haller MJ, Atkinson MA. 2007. Assessing theIn VitroSuppressive Capacity of Regulatory T Cells. Immunological Investigations. 36(5-6):607-628. http://dx.doi.org/10.1080/08820130701790368