, Paisley, UK) A cell count was then performed using a haemocyto

, Paisley, UK). A cell count was then performed using a haemocytometer, and the cell morphology was examined on cytocentrifuge preparations stained with May Grunwald Giemsa. The purity of PMs was further determined using non-specific esterase staining [16], and the cell viability was determined by trypan blue dye exclusion. At this stage the cell suspension was then kept at 0 °C until assayed. C. albicans (Sigma Chemicals, Poole, UK) was heat killed by placing it in a hot bath at 60 °C for one hour. The yeast particles were then re-suspended and counted before labelling them with fluorescein isothiocyanate (FITC) (Sigma Chemicals, Poole, UK) according to

the method of Ragsdale and Grasso, with some modification [17]. Labelled yeast cells were LDN-193189 research buy LBH589 in vitro re-suspended at 5×107 cells/mL in 20% DMEM-FCS (Life Technologies, Paisley, UK) stored in a light-protected environment at 4 °C. This stock was used within ten weeks

of preparation. Yeast particles opsonisation was performed with pooled human serum (PHS) collected from healthy laboratory staff personnel and stored at −70 °C [18]. The opsonisation step was done one hour before the phagocytosis assay. PMs, which were prepared (1.3.3), were seeded at 0.5–1×106 cells/well in 24-well tissue culture plates (Nunclon, InterMed, Roskilde, Denmark) in RPMI/FCS at 37 °C and 5% CO2 air for two hours to allow for cell adherence. Non-adherent cells were thereafter gently washed out twice with warm HBSS (Life Technologies, Paisley, UK). The labelled

yeast particles were added to wells at a macrophage-to-particles ratio of 1:40 and phagocytosis Mirabegron was allowed to proceed for thirty minutes. In experiments in which GM-CSF was used, the cells were incubated with this cytokine at concentrations of 50, 100 or 500 IU/mL for thirty-six hours, and then phagocytosis was measured [19]. All samples were assayed in duplicates. At the end of this period, the plates were placed on ice and washed twice with ice-cold HBSS, as described previously [17]. Each wash involved gentle pipetting up and down ten times after which the supernatant was discarded. The plates were examined using an inverted microscope between washings to determine the number of internalised particles and to ensure that non-internalised particles, and not cells, were actually washed away. To ensure that there were no free or non-internalised particles left behind, the wells were rinsed once more with the same buffer in some experiments, and the fluorescence of the supernatant was measured using a spectrofluorometer (RF-540 Shimadzu Corp, Kyoto, Japan) at an excitation wavelength of 482 nm and an emission wavelength of 520 nm [17]. Finally cells were lysed using 0.1 N NaOH, and the fluorescence of the liberated intracellular particles in the supernatant was measured using spectrofluorometer (1.3.4).

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