Ph rodo red staphylococcus aureus bioparticles

The assessment of phagocytosis was performed using pH-rodo™ red Staphylococcus aureus bioparticles (Thermo Fisher Scientific, Roskilde, Denmark), which allow the visualization and quantification of ingested bioparticles. The bioparticles are nonfluorescent at neutral pH outside of the cell, but as they are internalized in endosomes that fuse with lysosomes, the pH gradually decreases, and their fluorescence increases.
After 6, 24 and 48 h of stimulation with bacterial supernatants and controls, as described previously, the media was discarded, and 50 µL pH-rodo BioParticles solution in Live Cell Imaging Solution (Invitrogen, Waltham, Massachusetts, USA) was added. Following a 2 h incubation at 37°C, fluorescence was measured at 544/590 nm (ex/em) using a plate reader. Next, cell viability was analyzed by adding 50 µL of 1 µM calcein AM to each well. Calcein AM is hydrolyzed intracellularly by esterases in viable cells, producing calcein, a strongly fluorescent compound. After 30 min of incubation at 37°C, fluorescence was measured at 490/520 nm (ex/em). The fluorescence intensity from the pH-rodo bioparticles was normalized to calcein fluorescence to account for the cytotoxic effects of the different stimuli. Three separate experiments were performed, each with seven technical replicates.

For isolation of CD11b⁺ cells of Gl261 tumors from ICB R, ICB NR, and C mice, myelin was removed of tumor single-cell suspension with myelin removal beads II (Miltenyi Biotec; 130-096) according to the manufacturer’s instruction. Subsequently, CD11b⁺ cells were purified using MagniSort™ Mouse CD11b Positive Selection Kit (eBioscience; 8802-6860-74). Ex vivo phagocytosis of CD11b⁺ cells was assessed as previously described^{22 (link)}. In brief, CD11b⁺ cells were plated onto ultra-low attachment 96-well plates (Corning) and incubated at 37 °C, 5% CO₂ for 20 min to allow for cell resting. CD11b⁺ cells were subsequently cultured at 37 °C, 5% CO₂ for 2 h with pHrodo™-red Staphylococcusaureus BioParticles (Thermo Fisher) according to the manufacturer’s instruction. Phagocytosis was assessed by flow cytometry analysis for pHrodo-red⁺ cells of macrophages (CD45^highCD11b⁺ cells) and microglia (CD45^lowCD11b⁺ cells). PD-L1 was blocked during incubation with pHrodo™-red S. aureus BioParticles with 20 µg ml⁻¹ anti-PD-L1 (10 F.9G2; BioXCell).