Whole Blood Measurement of Histone Modifications Linked to the Epigenetic Regulation of Gene Expression

Maria Watson1, David Hedley1

1 Ontario Cancer Institute/Princess Margaret Cancer Centre, Toronto, Ontario
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 6.36
DOI:  10.1002/0471142956.cy0636s71
Online Posting Date:  January, 2015
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Rapid progress is being made to understand the regulatory mechanisms that underlie the epigenetic control of gene expression through histone modification. It is now recognized that this plays a major role in normal development and disease. This unit describes the application of flow cytometry to the study of epigenetic mechanisms by combining labeling of individual histone modifications and phenotypic markers, and it also discusses practical issues to optimize staining. The focus is on normal blood and samples from leukemia patients, but it can also be applied to cells grown in tissue culture. © 2015 by John Wiley & Sons, Inc.

Keywords: epigenetics; histone; histone modifications; leukemia; drug treatment

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Table of Contents

  • Introduction
  • Basic Protocol 1: Flow Epigenetics in Whole Blood Samples
  • Alternate Protocol 1: Surface Marker Staining Prior to Fixation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Flow Epigenetics in Whole Blood Samples

  • Blood sample
  • Heparin sample tubes
  • 10% formaldehyde solution (Polysciences, cat. no. 04018)
  • Permeabilization buffer (see recipe)
  • Wash buffer (see recipe)
  • 2% fixative solution (see recipe)
  • Freezing medium (see recipe)
  • Histone antibodies (see Table 6.36.1)
  • Antibody diluent (Dako, cat. no. 50809)
  • Surface markers (see Table 6.36.1)
  • Run buffer (see recipe)
  • 75 × 15–mm polystyrene tubes
  • 37°C water bath
  • Centrifuge
  • Aspirator
  • Vortex
  • Flow cytometer
Table 6.6.1   MaterialsAntibodies Tested with Procedure

Antibody Supplier Cat. no. Clone no. Used at (/test)
Tri‐methyl‐histone H3 (Lys 27) Alexa 647 Cell Signaling Technology 12158BF C36B11 25 ng
Di‐methyl‐histone H3 (Lys 79) rabbit Cell Signaling Technology 5427 D15E8 100 ng
Goat‐anti rabbit Alexa 488 Molecular Probes A11034 N/A 20 ng
Acetylated‐histone H3 (lys 9) Pacific Blue Cell Signaling Technology 11857BC C5B11 100 ng
Di‐methyl‐histone H3 (Lys 9) mouse Active Motif 39683 MABI 0307 100 ng
Goat‐anti mouse Alexa 647 Molecular Probes A21240 N/A 20 ng
CD45 KrO Beckman Coulter PN A96416 J.33 1 μl
CD34 PC7 Beckman Coulter PN A51077 581 3 μl
CD14 PC7 Beckman Coulter PN A22331 RMO52 3 μl
CD3 PC7 Beckman Coulter 6607100 UCHT1 3 μl
CD4 FITC Beckman Coulter PN IM0448U 13B8.2 6 μl
CD117 PC5.5 Beckman Coulter PN A66333 104D2D1 3 μl

Alternate Protocol 1: Surface Marker Staining Prior to Fixation

  Additional Materials (also see protocol 1Basic Protocol)
  • 10× PBS
  • Rotating device
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Literature Cited

Literature Cited
  Arrowsmith, C.H., Bountra, C., Fish, P.V., Lee, K., and Schapira, M. 2012. Epigenetic protein families: A new frontier for drug discovery. Nat. Rev. Drug Discov. 11:384‐400.
  Behbahani, T.E., Kahl, P., von der Gathen, J., Heukamp, L.C., Baumann, C., Gütgemann, I., Walter, B., Hofstädter, F., Bastian, P.J., von Ruecker, A., Müller, S.C., Rogenhofer, S., and Ellinger, J. 2012. Alterations of global histone H4K20 methylation during prostate carcinogenesis. BMC Urol. 12:5.
  Chow, S., Hedley, D., Grom, P., Magari, R., Jacobberger, J.W., and Shankey, T.V. 2005. Whole blood fixation and permeabilization protocol with red blood cell lysis for flow cytometry of intracellular phosphorylated epitopes in leukocyte subpopulations. Cytometry A 67:4‐17.
  Fischle, W., Tseng, B.S., Dormann, H.L., Ueberheide, B.M., Garcia, B.A., Shabanowitz, J., Hunt, D.F., Funabiki, H., and Allis, C.D. 2005. Regulation of HP1‐chromatin binding by histone H3 methylation and phosphorylation. Nature 438:1116‐1122.
  Obier, N. and Muller, A.M. 2010. Chromatin flow cytometry identifies changes in epigenetic cell states. Cells Tissues Organs 191:167‐174.
  Smith, S.M., Kimyon, R.S., and Watters, J.J. 2014. Cell‐type‐specific Jumonji histone demethylase gene expression in the healthy rat CNS: Detection by a novel flow cytometry method. ASN Neuro 6:193‐207.
  Swiezewski, S., Liu, F., Magusin, A., and Dean, C. 2009. Cold‐induced silencing by long antisense transcripts of an Arabidopsis Polycomb target. Nature 462:799‐802.
  Watanabe, T., Morinaga, S., Akaike, M., Numata, M., Tamagawa, H., Yamamoto, N., Shiozawa, M., Ohkawa, S., Kameda, Y., Nakamura, Y., and Miyagi, Y. 2012. The cellular level of histone H3 lysine 4 dimethylation correlates with response to adjuvant gemcitabine in Japanese pancreatic cancer patients treated with surgery. Eur. J. Surg. Oncol. 38:1051‐1057.
  Watson, M., Chow, S., Baryste, D., Arrowsmith, C.H., Minden, M.D., and Hedley, D.W. 2013. The study of epigenetic mechanisms based on the analysis of histone modification patterns by flow cytometry. Cytometry A 85:78‐87.
  Wei, Y., Xia, W., Zhang, Z., Liu, J., Wang, H., Adsay, N.V., Albarracin, C., Yu, D., Abbruzzese, J.L., Mills, G.B., Bast, R.C. Jr., Hortobagyi, G.N., and Hung, M.C. 2008. Loss of trimethylation at lysine 27 of histone H3 is a predictor of poor outcome in breast, ovarian, and pancreatic cancers. Mol. Carcinog. 47:701‐706.
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