Publication protocol
The canine macrophage-like cell line, DH82, was obtained from the American Type Culture Collection (ATCC, CLR-10389), propagated and maintained in Eagle's Minimum Essential Medium (EMEM, Quality Biological Inc., # 112-018-101) containing 15% heat-inactivated fetal bovine serum (Sigma Aldrich, F4135) and 1% penicillin-streptomycin-amphotericin-B mixture (Lonza, # 17–602E). Cells were grown to confluency, trypsinized and seeded into 24-well Costar tissue culture plates (Corning Inc., CLS3527) at a density of 1 × 106 cells per well and allowed to adhere. pHrodo™ Red-labeled bacteria were added at a ratio of 1:100 (macrophage: bacteria) and incubated for 30 minutes at 37°C to facilitate phagocytosis. A control plate with the same components was incubated on ice for the same duration. After incubation, phagocytosis was stopped by the addition of ice cold PBS containing 5 mM EDTA. The plate was allowed to sit for 5 minutes at room temperature. The PBS-EDTA was gently aspirated and the adherent cells were harvested by standard trypsin treatment for 5 minutes at 37°C. The contents from each well were transferred into a microcentrifuge tube and centrifuged at 5000xg for 10 minutes. Cells were washed twice and suspended in 1 ml of PBS and analyzed by flow cytometry. Each sample was run in triplicate and analyzed individually. Non-infected cells served as negative control to set the cut-off for the discrimination of pHrodo™ Red-negative and -positive cells, and the cells incubated on ice served as control to determine the percentage of phagocytosis.
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