Lionheart fx automated microscope

A549 cells were seeded in 12-well plates at a density of 3.5 × 10⁴ cells per well. After 24 h, cells were transfected in triplicate with plasmids (GFP or OAS1) using Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific). Live-cell imaging was performed using the Lionheart FX Automated Microscope (BioTek) equipped with full temperature and CO₂ control to maintain 37 °C and 5% CO₂. Images were collected using a ×4 magnification right after transfection and then at 24, 48, 72 and 96 h after transfection. Data were processed with Gen5 Image+ software (BioTek) to determine cell counts and are presented normalized to cells transfected with GFP.

The dorsal skin sample from each mouse was fixed in 4% paraformaldehyde and embedded in paraffin. The sections were cut at 5 μm and stained with hematoxylin and eosin (H&E). Histological analysis was performed using a Lionheart FX Automated Microscope and Gen5 Imager software (BioTek Instruments Inc., Winooski, VT, USA).

MEF cells were first trypsinized and replated onto fibrinogen-coated µ-slide 8-well plates (ibidi 80826) at a density of ~10,000 cells per well with a complete medium. Time-lapse images were acquired using BioTek Lionheart FX Automated Microscope with 20× phase lens at 10 min intervals of ~18 hr at 37°C with 5% CO₂. The velocity of MEFs was measured using ImageJ with the MTrackJ plugin and plotted in Prism 6.0 (GraphPad). The rose plots and directionality index were generated or calculated by the Chemotaxis and Migration Tool 2.0.

Mitochondrial membrane potential in live cells was assessed using the TMRE probe, as previously described^{41 (link)}. Cells were seeded in 96 well plates and treated with compound of interest for 72 h. Cells were then treated with 1 µM TMRE for 20 min at 37 °C. The cells were then washed with PBS containing 0.2% bovine serum albumin (BSA) and fluorescence was measured with a microplate reader (λ_ex = 549 nm, λ_em = 575 nm; SpectraMax, Molecular Devices, CA, USA). For live cell imaging, cells were seeded in 96-well clear bottom black polystyrene microplates (Corning™, catalog no. 07-200-565) at a density of 5 × 10³ cells and treated with compound of interest for 72 h. Cells were then treated with 200 nM TMRE for 20 min at 37 °C, washed with 0.2% BSA in PBS and imaged and quantified TMRE stained cells with a Lionheart FX automated microscope (BioTek, VT, USA). Fluorescence was measured with an excitation of 549 nm and an emission of 575 nm (Texas-Red filter). In order to account for variations in cell location in the well the fluorescence was measured with a 2 × 2 area scan and the results averaged. The mean objective intensity was measured and normalized by cell counting in each well using Gen5TM 3.0 software (BioTek, VT, USA). Data is represented as the mean of 9 determinations.

At 6, 48, or 72 hours post-infection (HPI), infected mice were euthanized and lungs (6 HPI), lungs and spleen (48 HPI), or lungs, spleen, liver, and blood (72 HPI) were collected. Trachea and other respiratory tissue were not harvested. Tissues homogenized in sterile PBS were serially diluted and plated in duplicate on HIA for bacterial enumeration. Serum was isolated following blood collection and analyzed by a multiplex cytokine assay (Millipore Sigma, Burlington, MA) for 5 pro-inflammatory cytokines known to play a role in plague: IFNγ, TNFα, IL1β, IL6, and IL10 [27 , 28 ]. Moribund mice and those found dead were necropsied and lungs, liver, and spleens were fixed in 10% formalin and stained with hematoxylin and eosin. For quantification of pathological lesions, sample identities were blinded for analysis. Severity scoring (0–4) was based on the size and frequency of necrotic and inflammatory lesions, with 4 being the most severe. Imaging of whole lung was performed on a Lionheart FX automated microscope (Biotek, Winooski, VT) at 4X magnification in montage mode. Stitching was performed without compression in post processing.

U937 cells were plated (10,000 cells/well) on a 96-well glass bottom dish (Cellvis, Mountain View, CA, USA) coated with Type I collagen and treated with 100 µg/mL of respective SE. The plate was centrifuged at 200× g for 8 min to facilitate cellular adherence to the bottom of the well and incubated at 37 °C for 18 h. Following incubation, cells were washed with PBS and fixed with 4% paraformaldehyde (PFA) in PBS for 15 min. Cells were then permeabilized by incubation in 0.1% TritonX-100 for 10 min. AlexaFluor 594 Phalloidin (Thermofisher, Grand Island, NY, USA) and Alexa Fluor 488 Vinculin (Thermofisher, Grand Island, NY, USA) were applied in a 1:40 dilution for 1 h, followed by a 5-min DAPI stain. Images were acquired using a Lionheart FX Automated Microscope (Biotek, Winooski, VT, USA). Representative 10× and 60× images were acquired manually for five fields of view per well. Image procession was performed using Gen5 ImagePrime. Quantification of cellular size, area, and circularity was performed by Gen5 ImagePrime via masking of phalloidin. A circularity metric was created by inputting the equation $C=4\pi A/P^2$ , where $C$ is the circularity, $A$ is the area of the cell, and $P$ is the perimeter of the cell (https://imagej.nih.gov/ij/plugins/circularity.html).