Nunc 8 well chamber slides

APC (BMDC, BMDC+SL8 or sorted TAM) were obtained from mTmG mice, and previously activated OT-I T cells were stained for 15 minutes at 37°C with 2 μM CFSE (Invitrogen) in PBS and washed in RMPI prior to use. Cells were co-cultured in NUNC 8 well chamber slides (Thermo Scientific) that were coated with fibronectin (2μg/ml; EMD Millipore) in PBS at 37°C for 1 hour before use. APCs in phenol red-free RPMI were allowed to attach to the chamber slides for 20–30 minutes at 37°C and 5% CO₂. Right before imaging, T cells (resuspended in 0.1% agarose) were added to the wells and slides were loaded for imaging. To visualize the interaction between different APC populations and T cells, a conventional widefield Zeiss Axiovert 200M was used with a Sutter Lambda XL illumination source, running on μMagellan software. Images were acquired every 2 minutes for 6 hours using a 20x objective. Samples were kept at 37°C using a heated robotic stage. Image analysis was performed in Imaris (Bitplane) and ImageJ.