Phrodo red staphylococcus aureus bioparticles conjugate for phagocytosis

Biological particulate matter (PM), which is derived from pHrodo™ Red Staphylococcus aureus Bioparticles™ Conjugate for phagocytosis (A10010), was purchased from Invitrogen (Woltham, MA, USA). Oligomycin (ab141829), the PH domain inhibitor MiTMAB (ab120466) and OctMAB (ab120467) were purchased from Abcam (Cambridge, UK). DPI (81050) was purchased from Cayman Chemical Co. (Ann Arbor, Michigan, USA). PerCP Cy5.5-conjugated anti-human CD11b (65–0112-U100) was purchased from Tombo Biosciences (San Diego, CA, USA). Anti-mouse Ly6G particles (558111) were purchased from BD Biosciences (San Jose, CA, USA). Adenosine triphosphate ATP (017-21511) was purchased from Wako (Osaka, Japan). The IKK inhibitor VII (401486) was purchased from Calbiochem (Darmstadt, Germany). BAY 11-7085 (BML-EI279) was purchased from Enzo Life Sciences (Farmingdale, NY, USA). LY294002 (#9901S) was purchased from Cell Signaling Technology (Danvers, MA, USA). These inhibitors were dissolved in dimethyl sulfoxide (DMSO) and diluted in PBS before treatment. Bortezomib (7282) was purchased from Tocris (Minneapolis, MN, USA). MG-132 (474790) was purchased from Calbiochem. Bortezomib and MG-132 were dissolved in ethanol and diluted in PBS before treatment.

Isolated neutrophils were cultured as described above. Instead of applying the CM-H₂DCFDA, here, pHrodo™ Red Staphylococcus aureus Bioparticles® Conjugate for Phagocytosis (Invitrogen, Darmstadt) were used. 100 μl pHrodo™ Red Staphylococcus aureus Bioparticles® were added to each sample and the samples were incubated for 1 hour at 37 °C and 5% CO₂. Thereafter, cells were washed with FACS buffer, the supernatants were removed, and cells were resuspended in 200 μl FACS buffer for subsequent flow cytometric analyses as described above. The gating was performed as indicated for ROS production in Fig. 3 a with the difference that phagocytosis negative CD16⁺ cells were applied for the settings. The percentage of positive cells for phagocytosis and the MFU were determined.