FIG 2.

Imaging flow cytometry-based quantification of calnexin-GFP on LCVs in homogenized amoebae. D. discoideum Ax3 dually producing AmtA-mCherry and calnexin-GFP (pAW16) were infected (MOI, 50) for up to 2 h with mPlum-producing virulent L. pneumophila JR32 or ΔicmT(pAW14). After homogenization and fixation, 20,000 events were acquired with an imaging flow cytometer. The analysis was performed in 6 steps, where the cells gated in one step were carried on to the subsequent step. Examples of included and excluded cells are shown for each step (arrows). (i) Events in focus were gated using the feature Gradient RMS_M05_Lpn. (ii) Intact cells and large membrane aggregates were excluded using the feature Area_MC. (iii) Events displaying a sufficiently high mPlum signal were gated using Max Pixel_MC_Lpn. (iv) LCVs containing one bacterium only were selected using the features Spot Count_Spot(M05, Lpn, Bright, 8.5, 1, 0) versus Area_Object(M05, Lpn, Tight). (v) AmtA-mCherry-positive events with the AmtA signal colocalizing with the mPlum signal were gated using the features Max Pixel_MC_AmtA versus Bright Detail Similarity R3_MC_Lpn_AmtA. (vi) Finally, in the gated AmtA-positive LCVs, the intensity of calnexin-GFP was quantified using Intensity_MC_Cnx. (Left) The histogram shows an overlay of JR32-containing (green) and ΔicmT mutant-containing (red) LCVs at 2 h postinfection. (Right) Alternatively, the gated AmtA-positive LCVs were analyzed using Intensity_MC_Cnx versus Bright Detail Similarity R3_MC_Lpn_Cnx, yielding a percentage of LCVs positive for calnexin-GFP and with the GFP colocalizing with the bacterium. The dot plot shows an overlay of JR32-containing (green) and ΔicmT mutant-containing (red) LCVs at 2 h postinfection.