Mφ cultured at a density of 400,000/well (96-well plate) were transfected on day 7 with miScript viral miRNA or control miRNA mimics (Qiagen). After 24 h, phagocytosis assay was performed with pHrodo Red Escherichia coli bioparticles conjugate (Invitrogen), as previously described (26 (link)). Briefly, the labeled bioparticles were resuspended in Live Imaging Buffer (Life Technologies) and homogenized by sonication for 2 min. Culture media was replaced with resuspended labeled E. coli and incubated for 1 h.
Similar experiments were performed with U937 differentiated macrophages. HOK-derived exosomes were transfected with miR-K12-3-3p or control mimic as described above. After 24 h, cells were assayed for E. coli phagocytosis and incubated for 4 h. As a negative control, cells were treated with 5 mM cytochalasin D (Sigma-Aldrich) prior to adding bioparticles. The cells were washed three times with PBS, fixed with 4% PFA, and analyzed by flow cytometry. Images were captured using a Zeiss LSM 710 confocal microscope (Carl Zeiss, Oberkochen, Germany) with 403/1.2 Water DIC C-Apochromat objective and 2× zoom or EVOS florescent microscope (Life Technologies) at original magnification. Confocal images were processed on ZEN lite software. Images were captured for four independent, randomly selected fields for each donor.
Similar experiments were performed with U937 differentiated macrophages. HOK-derived exosomes were transfected with miR-K12-3-3p or control mimic as described above. After 24 h, cells were assayed for E. coli phagocytosis and incubated for 4 h. As a negative control, cells were treated with 5 mM cytochalasin D (Sigma-Aldrich) prior to adding bioparticles. The cells were washed three times with PBS, fixed with 4% PFA, and analyzed by flow cytometry. Images were captured using a Zeiss LSM 710 confocal microscope (Carl Zeiss, Oberkochen, Germany) with 403/1.2 Water DIC C-Apochromat objective and 2× zoom or EVOS florescent microscope (Life Technologies) at original magnification. Confocal images were processed on ZEN lite software. Images were captured for four independent, randomly selected fields for each donor.