Fluorescent beads

WT and KO ATDCs were pulsed with different doses of OVA-Alexa 647 for 15 min and then chased for 30 min at 37 or 4°C. To study phagocytosis, ATDCs were pulsed with fluorescent beads (Sigma) at different dilutions.

2 mL of degassed PDMS was poured and cured into each well of a 6-well plate (Figure 1C). Each PDMS well was punched along the shape of a 12 mm glass slide and each oxidized PAA-coated PDMS slide was set at the bottom of the well. Trypsinized cells were mixed with neutralized type I bovine collagen (BD Biosciences) to create a suspension of 10 million cells / mL in 2 mg/mL of collagen. For cell-free experiments, 1 µm diameter fluorescent beads (Sigma) were added to the neutralized collagen solution. 6 µL of the collagen gel solution was dispensed over each adhesive pattern and allowed to polymerize for 45 min in a humidified cell incubator (37 °C, 5% CO2). 250 µL of either 2.5 mg/mL neutralized collagen or 2.5 mg/mL neutralized collagen supplemented with growth factor reduced matrigel (BD Biosciences: 61% laminin, 30% collagen IV, 7% entacin, and 2% other proteins including proteoglycans) mixture of collagen gel and matrigel solutions (75v/v% collagen I + 25v/v% marigel) was poured on each well and allowed to polymerize for 1 hour in the humidified incubator. 2 mL of cell culture media were added to each well and cultured at 37 °C. For observation of non-specific cellular movement, Latrunculin (10µM) was added to the media in control samples to prevent actin polymerization and hence migration.