Measurement of Low‐Abundance Intracellular mRNA Using Amplified FISH Staining and Image‐Based Flow Cytometry

Andrea L. Henning1, Jill N. Best Sampson1, Brian Keith McFarlin1

1 Applied Physiology Laboratory, University of North Texas, Denton, Texas
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 7.46
DOI:  10.1002/0471142956.cy0746s76
Online Posting Date:  April, 2016
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Recent advances in instrument design and reagent development have enabled the rapid progression in available measurement techniques in the field of flow cytometry. In particular, image‐based flow cytometry extends the analysis capacity found in traditional flow cytometry. Until recently, it was not possible to measure intracellular mRNA in specific phenotypes of cells by flow cytometry. In this protocol, a method of completing simultaneous intracellular measurement of mRNA and protein for PPAR‐gamma in peripheral blood monocytes, which have been exposed in vitro to modified LDL, is described. The process of PPAR‐gamma activation following uptake of modified LDL is believed to play a role in the development of atherogenesis. PPAR‐gamma mRNA measurement was made possible using an amplified FISH technique (PrimeFlow RNA Assay) that allowed for detection of low‐abundant intracellular mRNA expression. This protocol represents a continued effort by the authors’ laboratory to establish and validate new techniques to assess the role of the immune system in chronic disease. © 2016 by John Wiley & Sons, Inc.

Keywords: image‐based flow cytometry; intracellular mRNA; PPAR‐gamma; FlowSight; primeflow; monocytes; modified LDL uptake

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Table of Contents

  • Introduction
  • Basic Protocol 1: Detection of Intracellular mRNA in Monocytes Following Exposure to Modified LDL
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Detection of Intracellular mRNA in Monocytes Following Exposure to Modified LDL

  • Isolated monocytes
  • RPMI 1640 (with phenol red, sodium bicarbonate, and L‐glutamine)
  • Activation factors (e.g., DiI AcLDL, ThermoFisher Scientific)
  • Phosphate‐buffered saline (PBS)
  • Cell surface antibodies (e.g., CD14‐APCeFluor780 and CD16‐BV605)
  • PrimeFlow RNA Assay Kit (Affymetrix, cat. no. 19361) containing:
  • Fixation buffer 1 (see recipe)
  • Permeabilization buffer (10×) (see recipe)
  • Intracellular antibodies (e.g., PPARγ‐Atto395)
  • Fixation buffer 2 (8×) (see recipe)
  • Wash buffer
  • Target probes, e.g., PPARγ type 4 probe (Alexa Fluor 488) and normalizer type 1probe (Alexa Fluor 647)
  • Positive control target probe set (20×)
  • Target probe diluent
  • PreAmp mix
  • Amp mix
  • Label probe diluent
  • RNA label probes (100×)
  • Storage buffer
  • Flow cytometry staining buffer
  • 1.2‐ml tubes
  • Breathable plate sealer (such as AeraSeal)
  • 37°C, 5% CO 2 incubator
  • Electronic pipet (Integra single channel pipet)
  • Centrifuge
  • Aluminum foil
  • Incubator validated to maintain 40° ± 1°C
  • 96‐well U‐bottom plates
  • Flow cytometer with at least two lasers: blue (488 nm) and red (633 nm) (FlowSight, EMD Millipore)
  • IDEAS software
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Literature Cited

Literature Cited
  Ferrara, L.A., Capaldo, B., Mancusi, C., Lee, E.T., Howard, B.V., Devereux, R.B., and de Simone, G. 2014. Cardiometabolic risk in overweight subjects with or without relative fat‐free mass deficiency: The strong heart study. Nutr. Metab. Cardiovasc. Dis. 24:271‐276. doi: 10.1016/j.numecd.2013.08.009.
  Henning, A.L., Venable, A.S., Prado, E.A., Best Sampson, J.N., and McFarlin, B.K. 2015. Using Image‐based flow cytometry to measure monocyte oxidized LDL phagocytosis: A potential risk factor for CVD. J. Immunol. Methods 423:78‐84. doi: 10.1016/j.jim.2015.03.015.
  Herieka, M. and Erridge, C. 2014. High‐fat meal induced postprandial inflammation. Mol. Nutr. Food Res. 58:136‐146. doi: 10.1002/mnfr.201300104.
  McFarlin, B.K., Williams, R.R., Venable, A.S., Dwyer, K.C., and Haviland, D.L. 2013. Image‐based cytometry reveals three distinct subsets of activated granulocytes based on phagocytosis and oxidative burst. Cytometry A 83:745‐751. doi: 10.1002/cyto.a.22330.
  McFarlin, B.K., Venable, A.S., Prado, E.A., Henning, A.L., and Williams, R.R. 2014. Image‐based flow cytometry technique to evaluate changes in granulocyte function in vitro. J. Vis. Exp.
  Venable, A.S., Williams, R.R., Haviland, D.L., and McFarlin, B.K. 2014. An analysis of endothelial microparticles as a function of cell surface antibodies and centrifugation techniques. J. Immunol. Methods 406:117‐123. doi: 10.1016/j.jim.2014.02.010.
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