Journal of Leukocyte Biology Volume 55, April 1994 461 Slow calcium waves imaged in myeloid cells derived from neonatal cord blood G. M. Roberts, E. V. Davies, and M. B. Hallett Department of Surgery, University of Wales College of Medicine, Heath Park, Cardiff United Kingdom Abstract: Myeloid cells were derived from neonatal cord blood by culture with granulocyte-macrophage colony- stimulating factor for approximately 8 days. The resultant cell population contained large adherent cells (diameter 90 jim), expressing formylated peptide receptors that were functionally coupled to cytosolic free Ca2 signaling. Imaging of the cytosolic free Ca2 changes in these cells revealed initial focal release of Ca2 from a site from within the cell, with elevated Ca2 also near the cell edge. Increased cytosolic free Ca2 moved as a slow oscillating wave across the cell (velocity 1 jim/s). As similar events may occur in mature neutrophils and monocytes but be difficult to resolve because of the small size of these cells, it was concluded that neonatal myeloid cells may provide a useful model system for the investigation of Ca2 signal- ing in myeloid cells. J. Leukoc. Biol. 55: 461-466; 1994. Key Words: signaling . calcium oscillations cakium imaging INTRODUCTION Cytosolic free Ca2 plays an important role in signaling cellu- lar events in myeloid cells. In both neutrophils and mono- cytes, elevation in cytosolic free Ca2 concentration signals some key cellular events such as activation of the NADPH oxidase, exocytosis, and chemotaxis [1-3]. The rise in cyto- solic free Ca2 concentration in neutrophils stimulated by f-Met-Leu-Phe (fMLP) occurs as a result of both transmem- brane influx of extracellular Ca2 and release of Ca2 from intracellular organelle stores. Although the two events can be dissociated, by either the use of Ca2 channel blockers [4, 5] or the absence of extraceliular Ca2 [6, 7], the spatial and temporal characteristics of these two events have not been visualized in neutrophils under physiological conditions. The possibility exists that adult neutrophils are too small to give resolvable separation of the two events in time or space. Neonatal cord blood contains immature cells that can differentiate into myeloid and dendritic cells [8-11]. The myeloid cells derived from neonatal cord blood are larger than mature neutrophils and thus may provide an opportu- nity for visualizing the spatial and temporal separation of transmembrane influx of Ca2 and release from intracellular stores during activation of the formylated peptide receptor. In this paper, we report experimental conditions that generate myeloid cells expressing formylated peptide recep- tors (FPRs] that are coupled to cytosolic Ca2 signaling. We demonstrate slow signaling events in these cells and clear visualization of both transmembrane influx and release of Ca2 from intracellular stores. These myeioid cells derived from neonatal cord blood may thus prove to be a useful model for investigating these two intracellular Ca2 events. MATERIALS AND METHODS Materials Fura-2/AM, pluronic F-127 and formyl-Nle-Leu-Phe-Tyr- Lys-fluorescein derivative were purchased from Molecular Probes, Eugene, OR; f-Met-Leu-Phe, GM-CSF, and lipo- poiysaccharide (LPS) from Sigma Chemicals, Poole, Dorset, UK; RPM! 1640 and fetal calf serum from Gibco, Paisley, Scotland; and [3H]thymidine (3.07 TBq/mmol) from Amer- sham, Cardiff, UK. A serum-free proprietary medium, DCCM, was supplied by Biological Industries, Glasgow, Scotland. Tumor necrosis factor a (TNF-a) was a kind gift from Dr. N. Matthews (University of Wales College of Medicine). Generation of myeloid cells from neonatal cord blood Umbilical cord blood of neonates delivered normally or by cesarian section, collected in heparinized containers, was diluted 1:1 with phosphate-buffered saline (PBS, pH 7.2) and centrifuged through Ficoll-Hypaque. The interface cells were collected, washed twice in PBS, and contaminating erythrocytes removed by hypotonic lysis. Myeloid cells (ad- justed to 2.5 x 106 ml1) were cultured in either RPM! 1640 containing 10% fetal calf serum or DCCM plus GM-CSF. Assessment of cell growth Incorporation of [3H]thymidine was determined after 20 h incubation of cells (5 x 10/200 jil) with 20 jiCi/ml [3H]thy- midine before precipitation with trichioroacetic acid (10%) and scintillation counting. The DNA content of the myeioid cells was estimated using a modified method as described by Labarca and Paigen [12] and Rago et al. [13]. The fluores- cence of Hoechst 33258 (20 jig/ml) in 10 mM Tris, 1 mM EDTA, 2 M NaC1 added to freeze-thawed cells was deter- mined at an emission wavelength of 458 nm (excitation 356 nm). Calf thymus DNA was used to calibrate the assay. Abbreviations: fMLP, f-Met-Leu-Phe; FPR, formylated peptide recep- tor; GM-CSF, granulocyte-macrophage colony-stimulating factor; LPS, lipopolysaccharide; NSE, nonspecific esterase; PBS, phosphate-buffered saline; TNF-a, tumor necrosis factor a. Reprint requests: M. B. Hallett, Department of Surgery, University of Wales College of Medicine, Heath Park, Cardiff, CF4 4XN, United Kingdom. Received October 20, 1993; accepted November 17, 1993.