Isolation of UCMC
Human umbilical cord blood was collected during normal deliveries of new-born infants at the Mayo Clinic, Methodist Campus. Heparin-treated umbilical cord blood from normal individuals were immediately layered over Histopaque-1077 (Sigma-Aldrich, St. Louis, MO), and the tubes containing the cord blood were centrifuged at 400×g for 30 min at room temperature to obtain a mononuclear cell fraction. After washing twice with phosphate-buffered saline (PBS) and 1% bovine calf serum (HyClone Laboratories Inc., Logan, UT), UCMC were suspended in RPMI-1640 medium (Celox Laboratories Inc., Cherry Hill, NJ) supplemented with 10% bovine calf serum, 2 mM L-glutamine (Sigma-Aldrich, St. Louis, MO), and 50 µg/mL gentamicin (pH 7.4; Sigma-Aldrich, St. Louis, MO). A part of the suspended cells was counted on haemocytometer using Randolph's stain. Isolated UCMC were washed and 2×106 cells/mL was resuspended in the medium.
Cell culture
Flat-bottomed 96-well cell culture plates (Corning Inc., Corning, NY) were coated with 100 mg/mL of hyaluronic acid (HA) in PBS (Sigma-Aldrich, St. Louis, MO) at 37°C for 3 h. UCMC (2×105 cells) were suspended in 200 µL of medium and cultured in the presence of 5 ng/mL of IL-5 (a gift from Schering-Plough Research Institute, Kenilworth, NJ) or medium alone at 37°C in 5% CO2. The cells suspended in the presence of IL-5 were also cultured with 1×10-9 M – 1×10-6 M of dexamethasone (Sigma-Aldrich, St. Louis, MO), 3×10-5 M – 1×10-3 M of lidocaine (Sigma-Aldrich, St. Louis, MO) or medium alone. Half the volume of culture medium was changed weekly. Four wells were utilized for evaluating the cell harvest, morphology, and granule proteins. All assays were carried out in duplicate.
Harvest and morphology of cultured cells
Viability and total number of cultured cells were determined by trypan blue exclusion, and the cells developed in the culture were characterized using Wright-Giemsa stain and cyanide-resistant peroxidase stain. The number of total cells and the cell components were counted weekly for 4 weeks.
Eosinophil granule protein content in cultured cells
To examine eosinophil differentiation, we analyse the contents of eosinophil granule proteins, including eosinophil-derived neurotoxin (EDN) and eosinophil peroxidase (EPX), in the lysates of cultured cells. After half the volume (100 µL) of medium supernatant was carefully removed from each well, 100 µL of 1% NP-40 (Sigma-Aldrich, St. Louis, MO)/0.01 N HCl was poured into the wells. The cell lysate was stored at -20°C until measurement of EDN and EPX.
The concentration of EDN in sample lysate was measured by radioimmunoassay (RIA). The RIA for EDN is a double-antibody competition assay in which radioiodinated EDN, rabbit anti-EDN antibody, and burro anti-rabbit IgG are used as described previously [21,22].
The concentration of EPX in sample lysate was measured by RIA [23], which was modified as follows. Before the assay, Immulon-4 96-well plates (Dynex Technologies Inc., Chantilly, VA) were coated overnight at 4°C with 100 µL of anti-human EPO antibody (5 µg/mL in PBS) and blocked with 200 µL of phosphate, protamine, foetal bovine serum, and EDTA (PPF-E) for 2 h at room temperature. After washing the wells with washing buffer (0.1 M PO4, pH 7.5; Tween 20, 10 mL/L), 100 µL of samples diluted with PPF-E or standard control (purified EPX from sera of hypereosinophilic syndrome patients) were added to the wells in duplicate and incubated overnight at 4°C. Next, wells were washed again with washing buffer. A second antibody, anti-human EPX antibody radiolabelled with I125, was added to the wells (50 ng/mL in PPF-E buffer, 100 µL/well) and incubated for 6 h at room temperature. The wells were then washed, and antibodies radiolabelled with I125 were counted in a gamma scintillation counter.
Statistical analysis
Data were represented as mean ± SE, and the statistical significance of the differences was assessed with paired nonparametric Wilcoxon signed-rank test.