Phrodo ifl green stp ester amine reactive dye

For pHrodo phagocytosis assays, Kp isolates were first heat-killed by incubation of bacteria at 60°C for 1 hour. Heat-killed Kp isolates were labeled with pHrodo iFL Green STP ester amine reactive dye (Invitrogen™, Thermo Fisher Scientific) according to the manufacturer’s instructions. Cell culture plates with macrophages were placed on ice for 5 minutes, after which pHrodo-labeled bacteria were added at a multiplicity of infection (MOI) of 10:1 (bacteria:macrophages). Plates were then centrifuged at 400 x g for 10 minutes at 4°C, and subsequently, macrophages and bacteria were incubated at 37°C for 1.5 hours to allow phagocytosis. After this, macrophages were collected in ice-cold PBS and the phagocytosis of Kp was analyzed directly on a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). Uptake of Kp was assessed based on pHrodo fluorescence and using uninfected cells as a reference to define pHrodo⁺ cells. Macrophages in negative control wells were incubated with cytochalasin D (10 μM) for 1 hour before the addition of Kp bacteria. The percentage of pHrodo⁺ cells across macrophages incubated with each of the clinical Kp isolates was normalized to fold change compared to macrophages incubated with the reference research strain ATCC 43816 (ATCC).