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 (Ca2+i) induced by chemoattractants in polymorphonuclear leukocytes (PMN) using the new fluorescent Ca2+ chelator Indo-1 AM. Increases in cytosolic Ca2+ were measured by flow cytometry. With this approach, 98% of PMN were found to respond to F-Met-Leu-Phe (FMLP) and leukotriene B4 (LTB4) stimulation. Although both substances induced a rapid and relatively homogeneous rise in Ca2+i, only FMLP gave a sustained Ca2+i rise, whereas that induced by LTB4 appeared transient. In addition, the combined use of wide-angle light scatter and electronic cell volume measurement allowed analysis of Ca2+i variations in the total peripheral blood cell population. The supernatant of a human fibrous histiocytoma cell line (GCT) was able to increase Ca2+ release in PMN. This activity may be ascribed to a new granulocytic activation factor, as neither human recombinant interleukin-1 alpha (IL-1 alpha), 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 Ca2+i rise in PMN.
Lopez
M
M
Unité de Cancérologie et Thérapeutique Expérimentales U. 119 INSERM, Marseille, France.
Olive
D
D
Mannoni
P
P
D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002614:Chelating Agents; D003600:Cytosol / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007211:Indoles; D007975:Leukotriene B4 / Q000494:pharmacology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism
D002614:Chelating Agents; D007211:Indoles; C059587:Indo-1 pentaacetoxymethyl ester; D007975:Leukotriene B4; D009240:N-Formylmethionine Leucyl-Phenylalanine; D002118:Calcium