Anti cd45 bv510 clone hi30

To determine the effect of fungal stimuli on viability of PBMCs, apoptosis and necrosis were assessed using Annexin-V and propidium iodide (PI) staining after 24 h of stimulation with the various fungal species. The cells were transferred to a v-bottom plate, washed with PBA (PBS pH 7.4, 1% w/v bovine serum albumin), and CD45 stained was stained using anti-CD45-BV510 (clone HI30; Biolegend) for 25 min at 4 °C in the dark. After washing twice with PBA, cells were stained with FITC-conjugated Annexin-V (Biovision) for 15 min at 4 °C in the dark in the presence of 5 mM CaCl₂. Immediately afterwards, PI (Invitrogen Molecular Probes) was added and the cells were incubated an additional 5 min at 4 °C in the dark. The cells were measured using a CytoFLEX cytometer (Beckman Coulter). Data analyses were performed in FlowJo (vX.0.7). First, CD45⁺ cells were gated to exclude fungal particles from the analysis. In the CD45⁺ population single cells were selected by subsequent SSC/FSC and FSC-H/FSC-A gating. The percentage of cells positive for only FITC-Annexin-V were designated as apoptotic and cells additionally positive for PI were designated as necrotic. Etoposide-treated (50 µM; Sigma-Aldrich) and heat-killed cells were used as positive controls for apoptosis and necrosis, respectively.