Bacteria conjugated to pHrodo™ show a very low fluorescent signal at the neutral pH present on the cell find more surface, but emit a bright red fluorescence in the acidic environment of phago-lysosomes. This level of discrimination eliminates washing and quenching
steps that are necessary with other non pH-dependent indicators of bacterial uptake. Moreover the fOPA here described takes advantage of the introduction of specific markers of HL-60 differentiation to neutrophils, which allow keeping under control the variability of effector cells. The method was evaluated for sensitivity and specificity, by testing a panel of sera from mice immunized with different GBS glycoconjugate vaccines against polysaccharide Ia. kOPA titers were compared with fOPA titers, and a confocal microscopy analysis was conducted to study bacterial localization inside neutrophils, VX-809 price in the presence or in the absence of specific antibodies and
complement. GBS strains 515 (serotype Ia) (Baker et al., 1982) and COH1 (serotype III) (Wessels et al., 1992) were used in this work. Bacteria were grown in Todd–Hewitt Broth (THB) to an optical density at 600 nm (OD600 nm) of 0.45. Ten percent glycerol was added to the culture before dispensing 1 ml aliquots in cryo-vials for flash freezing in a 95% ethanol-dry ice bath. Frozen cultures were kept at − 70 °C until use. OPAs were performed with rabbit and mouse sera. Rabbit sera were raised by immunizing one animal with three doses of monovalent CRM197-conjugated polysaccharide Ia, Ib and III in presence of aluminum hydroxide (Alum). Mouse sera were pooled from animals immunized with a GBS vaccine composed by polysaccharide Ia, Ib and III conjugated to CRM197, formulated with Alum or MF59 (Podda, 2001). Animal treatments were performed in compliance with the Italian laws and approved by the institutional review board (Animal Ethical Committee) of Novartis Vaccines and Diagnostics, Siena, Italy. Bacteria were grown in THB
Phosphatidylinositol diacylglycerol-lyase to OD600 nm = 0.6, washed twice with Phosphate Buffered Saline (PBS, pH 7.2–7.4,Gibco) and suspended in half volume of PBS-0.08% paraformaldehyde (PFA, Sigma). Cells were incubated at 37 °C for 30 min and kept at 2–8 °C in PBS-0.08%PFA. Immediately before labeling, cells were washed with PBS, suspended at 20 mg (wet weight)/ml using a freshly prepared 100 mM Sodium Hydrogen Carbonate solution pH 8.5 (Merck) and split into aliquots of 750 μl. A 10 mM stock solution of PHrodo™ Succinimidyl Ester (Invitrogen) in dimethyl sulfoxide (Sigma) was diluted in the bacterial suspension at a final concentration of 0.1 mM. Each sample was incubated for 45 min at room temperature in the dark and then added with 750 μl of Hank’s Balanced Salt Solution with Ca2 + and Mg2 + (HBSS, Gibco), then spin down with a bench top centrifuge for 60 s at 14,000 ×g. The supernatant was aspirated and the pellet suspended in HBSS and stored in the dark at 4 °C for two months.