Nucblue live cell stain readyprobes reagent

Cells were fixed in 4% paraformaldehyde, blocked in PBS/0.1% BSA/0.05% Tween for 1 h, and then incubated with anti-rabbit polyclonal AVPR2 antibody (PA5-75,409, RRID:AB_2719137, Thermo Fisher Scientific, Walthan, MA, USA) (1:100 dilution) and Hoescht 333,342 (NucBlue Live cell Stain Ready Probes reagent, R37605, Life Technologies, Carlsbad, CA, USA) overnight. The day after, cells were washed twice in PBS and stained with anti-Rabbit IgG Alexa Fluor488 secondary antibody for 1 h at room temperature in the dark. Finally, cells were washed and mounted in fluorescent Prolong Gold Antifade medium (P36930, Life Technologies, Carlsbad, CA, USA) for observation under an inverted fluorescence microscope (Leica Biosystems, Milan, Italy) equipped with a camera.

All samples were fixed in 4% paraformaldehyde overnight, permeabilized with 100% methanol for 2 min, and blocked in 5% BSA for 1 h at room temperature. Samples were incubated with primary antibodies against collagen I (Col I, 1:1,000) and collagen III (Col III, 1:1,000) diluted in a blocking buffer overnight at 4 °C. The samples were then stained with secondary antibodies (Alexa Fluor Registered 594 goat anti-mouse IgG [1:200] and Alexa Fluor Registered 488 goat anti-rabbit IgG [1:200]) for 1 h, and nuclei were stained with 1 μg/ml NucBlue Live Cell Stain ReadyProbes reagent (Thermo Fisher, Waltham, MA, USA) for 20 min. The samples were scanned using fluorescence inverted microscopy (Leica Microsystems, Wetzlar, Germany). Fluorescence intensity was measured using ImageJ 1.36b imaging software (National Institutes of Health, Bethesda, MD, USA).

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)).

In total, 5 × 10⁴ cells were seeded in 24-well plates (TPP) and treated with different MGO concentrations as described above. Microscope imaging was taken 24 and 48 h after treatment. The cells were stained with a propidium iodide solution (PI, 1.0 mg/mL, Sigma-Aldrich) and NucBlue Live Cell Stain ReadyProbes reagent (Thermo Fisher Scientific). The cells were washed with Dulbecco’s phosphate-buffered saline (DPBS, Gibco, Thermo Fisher Scientific) and covered with FluoroBriteTM DMEM (Gibco, Thermo Fisher Scientific) and imaged with a Keyence BZ-800E microscope (Keyence, Neu-Isenburg, Germany). The quantification of PI and DAPI stained cells was performed using the IndentifyPrimaryObjects function of the software CellProfiler (Version 4.2.4, Broad Institute, Cambridge, MA, USA).

Apoptotic thymocytes were stained with 2.5 µM DeepRed dye (Invitrogen, Carlsbad, CA, USA) for 24 h, while eRBCs were labeled with 4 μM of a PKH26 Red Fluorescent Cell Linker Kit according to the protocol provided by the manufacturer. Apoptotic thymocytes and eryptotic RBCs were added to 2 × 10⁵ C57BL/6 macrophages in a 1:5 macrophage/target cell ratio for 1 h; then, the remaining cells were washed away. Following phagocytosis, BMDMs were fixed by 1% paraformaldehyde and stained with NucBlue Live Cell Stain Ready Probes reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Pictures were taken on fluorescent microscope (FLoid™ Cell Imaging Station, ThermoFisher, Waltham, MA, USA).

The stain was prepared by mixing 1 mL of PBS with 2 µL of Nucleolar-ID green (Enzo) and 3 drops of NucBlue Live Cell Stain ReadyProbes reagent (Invitrogen). Cells cultured in 35 mm plates at 80% confluence were washed once with PBS (37 °C). A mixture of 250 μL of stain mix and 250 μL of complete media was added to each plate. Plates were incubated at 37 °C in a humidified incubator with 5% CO₂ for 15 min. Live cell nucleolar images were captured using a Nikon Eclipse Ti inverted microscope at 40× (Nikon). Eight fields were counted for each cell line. Experiments were repeated at least three times. Significance was determined using a t-test and all error bars indicate SEM.