Flow cytometric measurements of cytoplasmic calcium changes in human platelets

Abstract

Thrombin-induced stimulation of human platelets is accompanied by a dramatic increase in cytoplasmic calcium concentrations followed by a slow decrease. These changes are very rapid, are maximal by 10-15 s, and can be detected with probes such as Indo-1. Suspension studies using spectrofluorometry, which reflect a value which is the average of 3 x lo7 cells per ml, indicate a thrombin dosedependent increase in cytoplasmic calcium at doses up to 0.025 units per ml. We show here, using flow cytometry, that at less than halfsaturating thrombin doses only subpopulations of platelets rather than the entire sample are responding. The extent of these responses, however, still depends on thrombin concentration. When the thrombin doses are between half and fully saturating, one subpopulation responds fully (i.e., its extent of increase in cytoplasmic calcium concentration, [Ca++]i, is 100% of that seen at saturating thrombin concentrations) while the remaining platelets respond partially or not at all. There is thus evidence of positive cooperativity leading to disproportionate thrombin receptor occupancy on different subpopulations when platelets are subjected to subsaturating doses of thrombin. The existence of responding subpopulations may explain how the reported multiple stimulations of the same suspension of platelets at low thrombin doses occur.

A Novel Assay to Measure the Calcium Flux in Human Basophils: Effects of Chemokines and Nerve Growth Factor

“…Flow cytometry has previously been used to measure calcium flux in other cell types, including eosinophils, lymphocytes, natural killer cells, and platelets [15][16][17][18]. Here we describe that calcium flux can be detected also in basophils contained in mixed PBMC preparations and double stained with CD123 and HLA-DR. Until now, it was impossible to measure calcium flux in native basophils, as these cells are rare in blood, and conventional methods such as fluorescence spectrometry rely on large amounts of purified cells.…”

An imaging flow cytometry-based approach to measuring the spatiotemporal calcium mobilisation in activated T cells

“…We have tested different ways in which an ant-TCR stimulus could be introduced with slower activation kinetics such as CD3-coated microspheres (data not shown) or through conjugate formation with antigen presenting cells (Filby et al, 2007). However for cell types such as platelets where activation-induced Ca 2+ flux occurs within seconds (Davies et al, 1988) our IFC method may not be suitable. Our IFC-based method excelled was when we began to analyse the multispectral imagery output from the ISx system.…”

Standardization of the Flow Cytometric Determination of HLA Class I Antigens, 'Platelet-Specific' Glycoproteins and Activation Markers

“…The determination of 'platelet-specific' antigens and HLA-class I antigens by FC is an accurate and quick meth od [1][2][3][4][5][6]. Investigations were carried out to compare several preparation methods for the FC determination of platelet glycoproteins.…”

“…This is of interest for the diagnosis of inherited deficiencies of the expression of 'platelet-specific' glycoproteins like GPIIb/lIIa and GPlb, as has been demonstrated in Glanzmann's thrombasthenia and in Bemard-Soulier syndrome [1,3]. Furthermore, FC allows the performance of function al tests on platelets to investigate the state of activation that corresponds to the situation in vivo [4,5] or the capacity for activation in vitro [4,6].…”