Phrodo green zymosan bioparticles

Macrophage progenitors were cultured for 7 days in macrophage medium in a 96-well plate at a density of 1 × 10⁵ cells/well. Mature iMΦ were incubated with or without reconstituted pHrodo Green Zymosan Bioparticles (Cat# P35365; Invitrogen, Waltham, MA, USA) at 37 °C for 2 h. Cells were harvested and analyzed by flow cytometry. Zymosan-free cells were used as negative controls to set a threshold for measuring the percentage of positive cells (Wilgenburg et al., 2013 (link)).

Phagocytosis, the process by which cells engulf and eliminate foreign particles or pathogens, is a critical mechanism used by immune cells, particularly macrophages and dendritic cells. Assessing phagocytic activity serves as a valuable marker for immune cell function in the context of anticancer immunity due to its role in clearing cancer cells, debris, and promoting an anti-tumour immune response [30 (link),36 (link)]. Lipopolysaccharide (LPS) was used to induce phagocytosis in PBMCs. LPS (Sigma-Aldrich, Rehovot, Israel) was introduced into three wells of a 96-well dark plate, with each well receiving 10 µL of a 5 µg/mL solution. Negative controls were established in another three wells using 10 µL of LPS diluent. The final three wells acted as media controls and contained only RPMI and 10 µL of 5 µg/mL LPS without cells. After 2 h of incubation and activation of phagocytic function, the dark plate was subjected to centrifugation and the supernatant was then removed. Subsequently, 100 µL of pHrodo Green Zymosan Bioparticles (Invitrogen, Eugene, OR, USA) [37 ] was added to each well, followed by incubation at 37 °C for one hour. Fluorescence measurements were performed using a fluorimeter (Fluoroskan Ascent, Thermo Fisher Scientific, Waltham, MA, USA), with excitation set at 510 nm and detection at 538 nm.

hESC-RPE cells were exposed for 24 hours to purified FITC-labeled photoreceptor outer segments of pig (gift from Dr. E. Nandrot). After washing with PBS, cells were fixed in cold methanol and labelled with DAPI. Images were taken with LSM-800 confocal microscope (Zeiss). hESCs derived RPE cells were also exposed to pHrodo Green Zymosan Bioparticles (Thermo Fisher Scientific) overnight at 37 °C. These particles are pH-sensitive and become fluorescent after cell entry and phagosome formation. As a negative control, phagocytosis assays were performed at 4 °C to block the phagocytic process. Plates were then read using a microplate reader (Clariostar-BMG LABTECH) and values were normalized to DAPI intensities.