Analysis of cytosolic ionized calcium variation in polymorphonuclear leukocytes using flow cytometry and Indo‐1 AM

The present report describes the increase in cytosolic‐free calcium levels (Ca 2+ i) induced by chemoattractants in polymorphonuclear leukocytes (PMN) using the new fluorescent Ca 2+ chelator Indo‐1 AM. Increases in cytosolic Ca 2+ were measured by flow cytometry. With this approach, 98% of PMN were found to respond to F‐Met‐Leu‐Phe (FMLP) and leukotriene B 4 (LTB 4 ) stimulation. Although both substances induced a rapid and relatively homogeneous rise in Ca 2+ i, only FMLP gave a sustained Ca 2+ i rise, whereas that induced by LTB 4 appeared transient. In addition, the combined use of wide‐angle light scatter and electronic cell volume measurement allowed analysis of Ca 2+ i variations in the total peripheral blood cell population. The supernatant of a human fibrous histiocytoma cell line (GCT) was able to increase Ca 2+ release in PMN. This activity may be ascribed to a new granulocytic activation factor, as neither human recombinant interleukin‐1α (IL‐1α), granulocyte‐colony stimulating factor (G‐CSF), nor granulocyte‐macrophage‐colony stimulating factor (GM‐CSF), which were present in this supernatant, were able to induce a Ca 2+ i rise in PMN.