An increase in intracellular ionized calcium concentration ([Ca2+]i) is an early indicator of cell activation. Intracellular ionized calcium can be measured by flow cytometry using the fluorescent dye indo-1. Other measurements can be made simultaneously, such as cell surface molecule expression, allowing the study of calcium responses of cell subsets.
Dr. Lawrence Lum used this assay to investigate the signal transduction of peripheral blood mononuclear cell (PBMC) samples from bone marrow transplant patients (see reference below). The normal PBMC sample pictured in the 3-dimensional plot above indo-1 ratio on the x-axis (projected towards the viewer), time on the y-axis (near horizontal, projected slightly away), and cell number on the z-axis (vertical) was stimulated with phorbol myristate acetate (PMA), a strong ionophore. A projected histogram of the indo-1 ratio appears in red on a line parallel to the x-axis. The stimulation point can be seen as the gap between the two tall peaks on the y-axis, during which the sample was briefly removed from the cytometer for the addition of PMA. Shortly afterwards (under a minute) the indo-1 ratio rises dramatically, indicating a large increase in intracellular calcium ion concentration typical of PMA stimulation. The sample was measured over just a few minutes total. Although PC-LYSYS* was used to generate statistics, this plot was made with IsoContour*.
*PC-LYSYS is a trademark of BD Biosciences. IsoContour is a trademark of Verity Software House, Inc.
Jin N, Lum L, Van Buren E, Lerman S, Walker A, June C. Signal transduction by B and T cells early after bone marrow transplantation: B cell calcuim flux responses are intact whereas lack of CD4 cells accounts for impaired T cell responses. Bone Marrow Transplant. 16: 103-109, 1995.