Colocalization Analysis of Transformed Rickettsia

Cell-free WT and transformed R. monacensis cells were resuspended in complete medium and incubated with NucBlue Live Cell Stain ReadyProbes reagent (1: 50 dilution) (Thermo Fisher Scientific) in the dark for 30 min at room temperature. Fifty-microliter aliquots of cell-free R. monacensis were deposited onto microscope slides (Cytospin centrifuge; Thermo Fisher) at 200 rpm for 3 min. The slides were mounted with 3 μL 1× phosphate-buffered saline (PBS) and imaged on an Olympus BX61 DSU confocal microscope with a 60× objective via a double-wavelength filter (DAPI, excitation at 365 nm and emission at 480 nm; FITC, excitation at 495 nm and emission at 519 nm). Colocalization of fluorescence from gfp_uv (transformed R. monacensis) and NucBlue (rickettsial DNA) was analyzed by determining signal overlap for each of three random fields of view using Image Fiji (with the JaCoP plugin and Co-localization Threshold plugin), Pearson’s coefficient (PCC), and calculation of Manders’ colocalization coefficients (MCCs) (26 (link), 27 (link)).