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
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Abstract

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

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1:

  Materials
  • 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.
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Figures

Videos

Literature Cited

Literature Cited
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