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

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.