Live Cell Phagocytosis Assay

BMDM were plated on a 96 well glass bottom plate with #1.5 cover glass and a black frame (CellVis) at 1×10⁵ cells per well in I-10 media overnight. The cells were stained with 5 μM CellTracker™ Orange CMTMR Dye for 37 °C x 30 min then washed. Cells were washed and incubated in 150 μL I-10 media. 50 μL of a 1:31 dilution of single CPS-expressing B. theta strains that endogenously expressed GFP with or without 10 μg/mL of B. theta antibody was added to each well. B. theta strains expressing a single CPS and GFP were grown in a 2mL TYG culture at 37 °C overnight to mid log phase. Cultures were washed once and resuspended in PBS prior to adding to the assay. Four hours later, images were acquired in the same z-plane using an Olympus IX70 microscope with a 100x/1.4NA oil objective, a Yokogawa spinning-disk confocal scanning unit, and a Hamamatsu Orca Flash4 CMOS camera. Cells were maintained at 37 °C with 5.0% CO₂ in a Tokai Hit humidified chamber. Images were acquired with NIS-Elements AR software and the number of bacteria uptaken per BMDM was measured manually using Imaris software. A total of 150 individual cells were measured for each bacterial strain analyzed with 50 cells measured per experiment in 3 experiments.