Fig. S9.

Characterization of human colonic specimen and derived cells. Viability (A) and purity (B and C) of cells isolated from human colonic lamina propria were systematically assessed by flow cytometry before infection, using Live/Dead fluorescent dye and anti-human CD45, Epcam, CD3, and CD19 fluorophore-conjugated antibodies, respectively. Data from two independent donors are represented in orange and purple. Unstained cells are depicted in gray. (D) For imaging cytometry analysis, apoptotic, autofluorescent, and misshapen cells were excluded after selection of focused, single, live loaded cells (Fig. S5 A and B) and before quantification of targeting mechanisms (Fig. S5 C–E). Apoptotic cells (dying cells) were excluded from the analysis as those with high values in the Contrast (measuring the sharpness quality of the BF image) and the Side-Scatter intensity features of the BF image (Left). Cells with high autofluorescence in the DsRed image were removed by creating a threshold mask delineating the top 50% intensity pixels in this channel (Middle). Remaining misshapen cells were removed using the “find the best feature” procedure (41) that identified the Intensity and Modulation (measures the intensity range of an image, normalized between 0 and 1) features calculated on the BF channel (Right).