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.