Back thinned electron multiplying charge coupled device camera

BMDMs were plated as a monolayer at 4×10⁶ cells/well onto coverslips in a 6-well tissue culture plate. After 24 h, the monolayer was infected with Lm expressing RFP (DP-L5538) at an MOI of 0.01 for 1 h. Cells were washed three times with PBS and incubated at 37°C in RPMI containing 10% FBS, 10% L-929 conditioned medium and 50 μg/mL gentamycin. At 6 h post infection, the coverslips were imaged in 25 mM Hepes buffered RPMI containing 10% FBS and 50 μg/mL gentamycin using a Quorum spinning disk confocal microscope (Leica DMIRE2 inverted fluorescence microscope equipped with a Hamamatsu back-thinned electron multiplying charge-coupled device camera, Yokogawa spinning disc head, and Volocity 6 software). Coverslips with BMDM from control and TIM4^−/− mice were placed side by side on a dual chamber heated stage at 37°C. Over the course of 12 h, 36 μm z-stacks with a 2 μm step were taken every 15 min at ten foci of infection per coverslip. The channels for DIC and red fluorescence were acquired throughout the experiment. Image analysis was performed to measure the number of infected cells per infection foci during the course of the experiment.