Neutrophil isolation and neutrophil-microbe interactions
Neutrophils were isolated from healthy donor blood (Research Blood Components, LLC, Watertown, MA) using a negative selection kit (StemCell Technologies, Inc. Cambridge, MA) according to manufacturers instructions. Cells were stained with Hoeschst 33342 (Thermo Fisher), washed, and resuspended at 40 x 10^6 cells per mL for loading into the device. Devices were loaded with mono- or co-cultures of A. graevenitzii and S. aureus as described above. Following thorough washing of the main channel, neutrophils were then loaded and imaging commenced.
Image processing, data acquisition, quantification, and analysis
During the experiments, a glass-bottom 6-well plate (Micro Device Instruments, Avon, MA, USA) with microfluidic device was placed on a fully automated Nikon TiE microscope. The microscope was fitted with an incubator humidified and heated at 37°C. Images were acquired through 10x or 20x objectives in phase contrast. Growth of bacteria and bacteria movement were recorded using time-lapse imaging. Individual frames were recorded at an interval of 10 minutes at 10x, 20x, or 40x objectives for 24 hours. For detailed observations, images were also acquired every 10 or 30 seconds, using an oil-immersion 100x objective, for a minimum of 90 minutes. The experiments for this study were repeated up to ten times, including all control experiments. Time lapse image sequences were analyzed by FIJI (Fiji Is Just ImageJ, NIH). Results were plotted using Graphpad Prism V8.2.1 and Sigma Plot version 12. Error bars represent mean ± SEM.