μg pcDNA3-FLAG-PU.1 with 0.5 μg pCMV-GFP for overexpression of mouse PU.1 and 1μg pGFP-V-RS-shSCR, -shA, -shB and -shD for knock-down of mouse PU.1 were prepared with 2 μl of Lipofectamine 2000, incubated for 20 min at room temperature and added to each well. After 8 hours of incubation 1 ml of growth medium was added to each well and plates were incubated for 2 days. Then the medium was replaced with 500 μl of fresh medium, and 25 μg of bioparticles were added to cells for 3 hour incubation. Bioparticles uptake was verified with a fluorescent microscope; then the cells were collected with trypsin (Gibco #25200), washed with PBS once and re-suspended in 500 μl PBS with 1% BSA. Cells were kept on ice and phagocytic activity was analyzed on an LSR II flow cytometer (BD Biosciences). At least 30,000 events were collected in each experiment, gated on FSC-A/SSC-A and further on FSC-A/FSC-W dot plot to analyze populations of viable single cells. Data were quantified using FCS Express 5 (De Novo Software) and GraphPad Prism 7 (GraphPad Software). Cells pretreated with 2 μM Cytochalasin D for 30 minutes before and during the uptake of bioparticles were used as a negative
