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Fluorescence Approaches to Image and Quantify the Demarcation Membrane System in Living Megakaryocytes

  • Sangar Osman
  • Daniel Dalmay
  • Martyn Mahaut-SmithEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1812)

Abstract

The demarcation membrane system (DMS) develops to provide additional surface membrane for the process of platelet production. The DMS is an invagination of the plasma membrane that can extend throughout the extranuclear volume of mature megakaryocytes and its lumen is continuous with the extracellular solution. DMS ultrastructure in fixed samples has been extensively studied using transmission electron microscopy (TEM) and more recently with focused ion beam scanning EM. In addition, whole cell patch clamp membrane capacitance provides a direct measurement of DMS content in living megakaryocytes. However, fluorescence methods to image and quantify the DMS in living megakaryocytes provide several advantages. For example, confocal fluorescence microscopy is easier to use compared to EM or electrophysiological methods and the required equipment is more readily available. In addition, use of living cells avoids artifacts known to occur during the fixation, dehydration, or embedding steps used to prepare EM samples. Here we describe the use of styryl dyes such as FM 1–43 or di-8-ANEPPS and impermeant fluorescent indicators of the extracellular space as simple approaches for imaging and quantification of the DMS.

Key words

Megakaryocytes Demarcation membrane system Styryl dyes Megakaryopoiesis 

Notes

Acknowledgments

We are grateful to Dr. Kees Straatman of the Advanced Imaging Facility, Core Biotechnology Services, University of Leicester for discussion on imaging and microscopy. Work in the authors’ laboratory that led to the methods described in this chapter was funded by the Medical Research Council and the British Heart Foundation. S.O. was supported by the Kurdistan Regional Government Ministry of Higher Education and Scientific Research.

Supplementary material

Supplementary Video 1

A video showing the rapid time course of staining of the demarcation membrane system in a rat megakaryocyte following perfusion with FM 2-10 and rapid destaining following wash. (MP4 2731 kb)

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Sangar Osman
    • 1
  • Daniel Dalmay
    • 1
  • Martyn Mahaut-Smith
    • 1
    Email author
  1. 1.Department of Molecular and Cell BiologyLancaster Road, University of LeicesterLeicesterUK

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