BD CompBeads were stained as compensation controls for V450 anti-human CD11b and for FITC anti-human CD35, while pHrodo™ labeled bacteria were used as phycoerythrin (PE) fluorescence to calculate the compensation matrix. The compensation values were calculated automatically by DiVa™ software. The BD High Throughput Sampler (HTS) System was used to run the plate samples. A total of 10,000 events were collected from each sample gated on live cells. Forward scatter and Side scatter were acquired on a linear scale and fluorescence was acquired on a logarithmic scale. PE and fluorescein isothiocyanate (FITC) were excited using 488 nm laser and the emission of fluorescence was collected using

585/42 nm and 530/30 nm filters, respectively. V450 and LIVE/DEAD

Fixable Aqua were excited by 405 laser and fluorescence emission was collected with 450/50 nm and 510/50 nm DF filters. After acquisition, all data were exported as Flow Cytometry Standard format 3.0 Ibrutinib mw files (FCS files) and analyzed by FlowJo (Mac-Version 9.1; Treestar US, Ashland, OR). Differentiated HL-60 cells were dispensed in 96 microtiter plates and incubated with labeled bacteria for 30 min in the presence of specific or unrelated serum and baby rabbit complement, under the same conditions and using the same concentration described for the fOPA. After incubation, cells were washed twice with PBS (centrifuging the plate at 900 rpm for 5 min at 2–8 °C) and fixed with 4% PFA in PBS for 5 min at 2–8 °C. After washing, Trametinib research buy bacteria were pelleted by centrifugation at 900 rpm for 5 min. The plasma membrane was then stained by incubating cells for 30 min at for 4 °C with 100 μl of Alexa Fluor 488-phalloidin (0.16 μM, Molecular Probes) solution or concanavalinA-FITC (Sigma) solution in PBS (2 μg/ml). After washing, cells were suspended in 10 μl of SlowFade Antifade kit (Molecular Probes) and mounted on a glass slide. Images were acquired on a Zeiss LSM 710 laser scanning confocal microscope. Each experiment was performed in triplicate. Data are represented as mean ± SD. Correlations were analyzed by a linear

regression model. Fitting was analyzed with the support of a statistical software (GraphPad Prism 5). The amine-reactive succinimidyl ester of pHrodo™ dye was used to label paraformaldehyde (PFA) fixed bacteria via amine groups present on the bacterial cell wall. To optimize bacterial labeling, PFA fixed bacteria were first incubated with 0.1 mM up to 0.9 mM concentrations of pHrodo™. A dye concentration of 0.1 mM, yielded the highest ratio between the mean fluorescence intensities of the positive and the negative controls (data not shown) was chosen for further use. To assess whether the fixation or conjugation steps altered the integrity of target antigens, labeled GBS Ia bacteria were compared with live bacteria for reactivity with a pool of mouse sera specific for polysaccharide Ia using flow cytometry analysis. As shown in Fig.