Phagocytosis Assays using Flow Cytometry

We also used two flow-cytometric assays of phagocytosis. First, 50 x 10⁶ AC were labeled with 75μg TAMRA (Invitrogen) in 1 ml PBS and 1 ml LCM for 15 min at 37°C (49 (link)–51 (link)). Labeled AC were washed two times with PBS to remove excess TAMRA. Second, S. aureus pre-labeled with pHrodo-green (Invitrogen) was opsonized by incubation in rabbit anti-staphylococcal mAb (Invitrogen) for 1 h in a 5% CO₂ environment at 37°C, then washed . Mø were plated at 3 x 10⁶ cells/well in 24-well plates. Mø pretreated with cytochalasin D (Sigma) for 1 h before exposure to florescent targets were used to establish baseline florescence. TAMRA-labeled AC or pHrodo-green labeled S. aureus targets were added to Mø for 1 h, then wells were washed briskly with cold PBS to remove unengulfed targets. Mø were detached from culture dishes using the dissociation enzyme TrypLE (Invitrogen), then centrifuged in flow tubes. Internalized fluorescent particles were detected by flow cytometry.