A correlation Sotrastaurin coefficient (R2) of 0.95 was obtained for the linear regression derived by plotting the dose dependent fluorescent signals measured for live and labeled bacteria. The data indicated that the integrity of target antigens was maintained after pHrodo™ labeling. Labeled bacteria were pre-incubated with heat inactivated rabbit serum specific for polysaccharide Ia, followed by HL-60 derived neutrophils and baby rabbit serum as source of complement, as described

in the Materials and methods section. To reduce assay variability, fluorescently labeled anti-CD35 and anti-CD11b antibodies were introduced as specific markers of HL-60 cell differentiation to phagocytes. Furthermore, the amine reactive dye LIVE/DEAD® was used to discriminate between live and dead HL-60 cells. This dye can permeate compromised membranes of necrotic cells and react with internal and surface exposed free amines, resulting in a more intense

fluorescent staining of dead cells compared to live cells where only surface free amines are available. After incubation, samples were analyzed by flow cytometry. Live HL-60 cells were first gated based on LIVE/DEAD® (Fig. 2A) and then based on forward scatter versus side scatter cytogram (Fig. 2B). The percentage of live cells shown in Fig. 2A was 79% of whole cells and this number varied from 72 to 85% in experiments performed in different days. Doublets were eliminated using SSC-W versus SSC-A plot (Fig. 2C). Moreover HL-60 positive to CD35 and CD11b receptors were gated to identify the neutrophil effector cell population (Fig. 2D), which corresponded to 62.5% of total live cells (from 45 to 78% BKM120 in the different experiments). Progesterone Finally, a phycoerythrin (PE) fluorescence histogram was used to

evaluate phagocytic activity, which was expressed as MFI and calculated by setting a Log 4 range over the whole scale in the PE channel (Fig. 2E). Focusing on effector cells allowed cleaning off, from the read out, the fluorescent signal of undifferentiated HL-60, as demonstrated by the disappearance in the immune serum histogram shown in Fig. 3A of the double peak present in Fig. 3B. In this way, enhanced assay sensitivity could be attained. We believe that the high variability in the number of live effector cells in the HL-60 population contributes to the low reproducibility encountered in the classical kOPA. This variability does not affect the phagocytic activity measurement of our fOPA method, as the fluorescent intensity derived from undifferentiated cells does not contribute to the read out of the assay. Indeed, the MFI values obtained for each dilution of a particular test serum were comparable irrespective of the proportion of live effector cells. Several assay conditions were tested to optimize the method: particularly, different bacteria to neutrophil ratios (Fig. 4), incubation times and complement concentrations were tested.