Hydrophilic PTFE cell culture inserts (0.4 μm diameter pores, 30 mm diameter, Merck Millipore Ltd) were placed in wells of 6-well culture plates. Arabidopsis cell suspension was cultured for 4 days to reach the exponential phase of growth. Twenty milliliters of Arabidopsis cells were washed and resuspended in the same volume of fresh supplemented Gamborg B5 medium. At time 0, a volume of 1.5 ml of Arabidopsis cells was placed in the apical side of the millicell and 30,000 conidia of B. cinerea were added to 1.5 ml supplemented Gamborg B5 medium in the basolateral compartment. In the mock condition, conidia were omitted. Culture plates were incubated at 22 °C under a 16 h (light)/8 h (dark) photoperiod with tridimensional shaking (50 rpm) and cells were collected by vacuum filtration at the indicated time points.
For neutral red staining, one volume of Arabidopsis cells was incubated with half volume of neutral red solution (0.1%) for 2 min and observed under light microscope.
For neutral red staining, one volume of Arabidopsis cells was incubated with half volume of neutral red solution (0.1%) for 2 min and observed under light microscope.