Measurement of T‐Cell Telomere Length Using Amplified‐Signal FISH Staining and Flow Cytometry

Andrea L. Henning1, Danielle E. Levitt1, Jakob L. Vingren1, Brian K. McFarlin1

1 Department of Biological Sciences, University of North Texas, Denton, Texas
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 7.47
DOI:  10.1002/cpcy.11
Online Posting Date:  January, 2017
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


Exposure to pathogen‐associated molecular patterns (PAMPS), damage‐associated molecular patterns (DAMPS), and physiologically challenging stimuli either positively or negatively affect leukocyte maturity. Cellular maturity has implications for the effectiveness of host response to bacterial or viral infection and/or tissue injury. Thus, the ability to accurately assess cellular maturity and health is important to fully understand immune status and function. The most common technique for measuring cellular maturity is to measure telomere length; however, existing techniques are not optimized for single‐cell measurements using flow cytometry. Specifically, existing methods used to measure telomere length are PCR‐based, making it difficult for a researcher to measure maturity within specific leukocyte subsets (e.g., T cells). In this report, we describe a new approach for the measurement of telomere length within individual T cells using an amplified fluorescence in situ hybridization (FISH) technique (PrimeFlow RNA Assay). The unique aspect of this technique is that it amplifies the fluorescent signal rather than the target up to 3000‐fold, resulting in the detection of as few as 1 copy of the target nucleic acid. While the current technique focuses on human T cells, this method can be broadly applied to a variety of cell types and disease models. © 2017 by John Wiley & Sons, Inc.

Keywords: senescence; cellular aging; disease risk; cell health

PDF or HTML at Wiley Online Library

Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
PDF or HTML at Wiley Online Library


Basic Protocol 1:

  • Target cells, e.g., peripheral blood mononuclear cells (PBMCs)
  • PBS (MilliporeSigma, cat. no. D5652)
  • Permeabilization buffer (see recipe)
  • 100× RNase Inhibitor 2 (Affymetrix eBioscience; PrimeFlow Reagent Kit)
  • 1000× RNase Inhibitor 1 (Affymetrix eBioscience; PrimeFlow Reagent Kit)
  • Fixation buffer 1 (see recipe)
  • Fixation buffer 2 (see recipe)
  • Wash buffer (Affymetrix eBioscience; PrimeFlow Reagent Kit)
  • Target probe diluent (Affymetrix eBioscience; PrimeFlow Reagent Kit)
  • PreAmp mix (Affymetrix eBioscience; PrimeFlow Reagent Kit)
  • Amp mix (Affymetrix eBioscience; PrimeFlow Reagent Kit)
  • Label probe diluent (Affymetrix eBioscience; PrimeFlow Reagent Kit)
  • Storage buffer (Affymetrix eBioscience; PrimeFlow Reagent Kit)
  • Flow cytometry staining buffer
  • 20× positive control target probe set (Affymetrix eBioscience)
  • 5× telomere target probes (Affymetrix eBioscience, e.g., AlexaFluor647)
  • 100× label probes (Affymetrix eBioscience; PrimeFlow Reagent Kit)
  • CD3‐PE‐Cy7 (Affymetrix eBioscience, cat. no. 25‐0038‐42)
  • CD4‐eFluor650 (Affymetrix eBioscience, cat. no. 48‐0048‐42)
  • CD8‐APCeFluor780 (Affymetrix eBioscience, cat. no. 47‐0149‐42)
  • CD45RA‐PE‐Cy5 (Affymetrix eBioscience, cat. no. 15‐0458‐42)
  • Viability Dye‐eFluor605 (Affymetrix eBioscience, cat. no. 65‐0866‐18)
  • Incubator (validated to maintain 40 ± 1°C)
  • 1.2‐ml polypropylene tubes
  • Flow cytometer with at least 2 lasers, blue (488 nm) and red (642 nm; MilliporeSigma EasyCyte 12HT)
NOTE: It is very important not to use antibodies labelled with PE‐Cy5.5; this tandem dye conjugation is not compatible with the DNA assay reagents described in this protocol.NOTE: This assay is highly temperature dependent. Ensure that the incubator holds a temperature of 40 ± 1°C. Also, the incubator must be capable of bringing the reaction mixture to 40 ± 1°C within 5 min of heating. There are several different incubators that work well for this function. It is ideal to use a metal heat block when possible to maintain consistent heat application. The incubator temperature should be precisely validated using a calibrated thermocouple. Failure to maintain temperature will result in inefficient amplification of the telomere signal and poor flow cytometry detection. Inability to amplify at the correct temperature is the biggest potential source of variability in this assay.
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
  Blackburn, E.H. 2000. Telomere states and cell fates. Nature 408:53‐56. doi: 10.1038/35040500.
  Budnar, R.G. Jr., Duplanty, A.A., Hill, D.W., McFarlin, B.K., and Vingren, J.L. 2014. The acute hormonal response to the kettlebell swing exercise. J. Strength Cond. Res. 28:2793‐2800. doi: 10.1519/JSC.0000000000000474.
  Carbonari, M., Cibati, M., Sette, N., Catizone, A., and Fiorilli, M. 2011. Measurement of telomere length using PNA probe by cytometry. Methods Cell Biol. 103:189‐202. doi: 10.1016/B978‐0‐12‐385493‐3.00008‐5.
  Carpenter, K.C., Breslin, W.L., Davidson, T., Adams, A., and McFarlin, B.K. 2013. Baker's yeast β‐glucan supplementation increases monocytes and cytokines post‐exercise: Implications for infection risk? Br. J. Nutr. 109:478‐486. doi: 10.1017/S0007114512001407.
  Cawthon, R.M. 2002. Telomere measurement by quantitative PCR. Nucleic Acids Res. 30:e47. doi: 10.1093/nar/30.10.e47.
  d'Adda di Fagagna, F., Reaper, P.M., Clay‐Farrace, L., Fiegler, H., Carr, P., Von Zglinicki, T., Saretzki, G., Carter, N.P., and Jackson, S.P. 2003. A DNA damage checkpoint response in telomere‐initiated senescence. Nature 426:194‐198. doi: 10.1038/nature02118.
  Effros, R.B. 2004. T cell replicative senescence: Pleiotropic effects on human aging. Ann. N. Y. Acad. Sci. 1019:123‐126. doi: 10.1196/annals.1297.022.
  Epel, E.S., Blackburn, E.H., Lin, J., Dhabhar, F.S., Adler, N.E., Morrow, J.D., and Cawthon, R.M. 2004. Accelerated telomere shortening in response to life stress. Proc. Natl. Acad. Sci. U.S.A. 101:17312‐17315. doi: 10.1073/pnas.0407162101.
  Fitzpatrick, A.L., Kronmal, R.A., Gardner, J.P., Psaty, B.M., Jenny, N.S., Tracy, R.P., Walston, J., Kimura, M., and Aviv, A. 2007. Leukocyte telomere length and cardiovascular disease in the cardiovascular health study. Am. J. Epidemiol. 165:14‐21. doi: 10.1093/aje/kwj346.
  Flores, I. and Blasco, M.A. 2010. The role of telomeres and telomerase in stem cell aging. FEBS Lett. 584:3826‐3830. doi: 10.1016/j.febslet.2010.07.042.
  Gil, M.E. and Coetzer, T.L. 2004. Real‐time quantitative PCR of telomere length. Mol. Biotechnol. 27:169‐172. doi: 10.1385/MB:27:2:169.
  Gutierrez‐Rodrigues, F., Santana‐Lemos, B.A., Scheucher, P.S., Alves‐Paiva, R.M., and Calado, R.T. 2014. Direct comparison of flow‐FISH and qPCR as diagnostic tests for telomere length measurement in humans. PLoS One 9:e113747. doi: 10.1371/journal.pone.0113747.
  Henning, A.L., Sampson, J.N., and McFarlin, B.K. 2016. Measurement of low‐abundance intracellular mRNA using amplified FISH staining and image‐based flow cytometry. Curr. Protoc. Cytom. 76:7.46.1‐7.46.8. doi: 10.1002/0471142956.cy0746s76.
  Levitt, D.E., Duplanty, A.A., Budnar, R.G. Jr., Luk, H.Y., Fernandez, A., Layman, T.J., Fancher, D.L., Hill, D.W., McFarlin, B.K., and Vingren, J.L. 2016. The effect of post‐resistance exercise alcohol ingestion on lipopolysaccharide‐stimulated cytokines. Eur. J. Appl. Physiol. 116:311‐318. doi: 10.1007/s00421‐015‐3278‐6.
  Lin, Y., Damjanovic, A., Metter, E.J., Nguyen, H., Truong, T., Najarro, K., Morris, C., Longo, D.L., Zhan, M., Ferrucci, L., Hodes, R.J., and Weng, N.P. 2015. Age‐associated telomere attrition of lymphocytes in vivo is co‐ordinated with changes in telomerase activity, composition of lymphocyte subsets and health conditions. Clin. Sci. 128:367‐377. doi: 10.1042/CS20140481.
  Maiti, S.N. 2016. Measurement of average telomere length in ex vivo expanded natural killer cells by fluorescence in situ hybridization (FISH) and flow cytometry. Methods Mol. Biol. 1441:57‐63. doi: 10.1007/978‐1‐4939‐3684‐7_5.
  McFarlin, B.K., Carpenter, K.C., Davidson, T., and McFarlin, M.A. 2013. Baker's yeast beta glucan supplementation increases salivary IgA and decreases cold/flu symptomatic days after intense exercise. J. Diet. Suppl. 10:171‐183. doi: 10.3109/19390211.2013.820248.
  McFarlin, B.K., Venable, A.S., Henning, A.L., Prado, E.A., Best Sampson, J.N., Vingren, J.L., and Hill, D.W. 2015a. Natural cocoa consumption: Potential to reduce atherogenic factors? J. Nutr. Biochem. 26:626‐632. doi: 10.1016/j.jnutbio.2014.12.015.
  McFarlin, B.K., Carpenter, K.C., Venable, A.S., Prado, E.A., and Henning, A.L. 2015b. Consumption of a high‐fat breakfast on consecutive days alters area‐under‐the‐curve for selected cardiovascular disease biomarkers. J. Mol. Pathophysiol. 4:6‐11. doi: 10.5455/jmp.20150127045909.
  Simon, N.M., Smoller, J.W., McNamara, K.L., Maser, R.S., Zalta, A.K., Pollack, M.H., Nierenberg, A.A., Fava, M., and Wong, K.K. 2006. Telomere shortening and mood disorders: Preliminary support for a chronic stress model of accelerated aging. Biol. Psychiatry 60:432‐435. doi: 10.1016/j.biopsych.2006.02.004.
  Strohacker, K., Breslin, W.L., Carpenter, K.C., Davidson, T.R., Agha, N.H., and McFarlin, B.K. 2012. Moderate‐intensity, premeal cycling blunts postprandial increases in monocyte cell surface CD18 and CD11a and endothelial microparticles following a high‐fat meal in young adults. Appl. Physiol. Nutr. Metab. 37:530‐539. doi: 10.1139/h2012‐034.
  von Zglinicki, T., Saretzki, G., Ladhoff, J., d'Adda di Fagagna, F., and Lackson, S.P. 2005. Human cell senescence as a DNA damage response. Mech. Aging Dev. 126:111‐117. doi: 10.1016/j.mad.2004.09.034.
  Willeit, P., Willeit, J., Brandstätter, A., Ehrlenbach, S., Mayr, A., Gasperi, A., Weger, S., Oberhollenzer, F., Reindl, M., Kronenberg, F., and Kiechl, S. 2010. Cellular aging reflected by leukocyte telomere length predicts advanced atherosclerosis and cardiovascular disease risk. Arterioscler. Thromb. Vasc. Biol. 30:1649‐1656. doi: 10.1161/ATVBAHA.110.205492.
  Wu, X., Amos, C.I., Zhu, Y., Zhao, H., Grossman, B.H., Shay, J.W., Luo, S., Hong, W.K., and Spitz, M.R. 2003. Telomere dysfunction: A potential cancer predisposition factor. J. Natl. Cancer Inst. 95:1211‐1218. doi: 10.1093/jnci/djg011.
PDF or HTML at Wiley Online Library