Hoescht stain

Following culture for 5 days, cells were washed with PBS, fixed with 4% formaldehyde, permeabilized with 0.2% Triton-X 100 and then blocked with 10% goat serum. To characterize cell shape, actin was stained with Texas-red phalloidin (Invitrogen) and nuclei were stained with Hoescht stain (Invitrogen). Images were acquired with a Nikon Eclipse (TiE) inverted fluorescence microscope at 20× magnification (PlanFluor 20X, 0.5 NA objective) with a CoolSNAP HQ2 Monochromatic CCD camera. Experiments were performed in triplicate, and images presented are representative from 5–10 random fields for each independent experiment. Images were quantified by determining the total intensity of phalloidin staining in Image J and then normalizing to the number of nuclei per image. To characterize circularity, area and perimeter of individual cells stained for actin were determined for each condition using Image J (NIH Freeware) image processing software, then circularity was determined using the equation circularity  = 4π(area/perimeter²). Three independent images were analyzed for each condition, and at least 10 cells were analyzed per image. Data is pooled from all 3 images analyzed per condition.

NHLF cells were grown in either 96-well Optilux plates (Falcon; 4,000 cells per well) or 384-well Optilix plates (2,500 cells per well) and allowed to attach overnight in complete FBM medium. Cells were then treated with compound for a specified period of time depending on experiment. Following compound treatment, HMOX1 protein was detected using indirect immunofluorescence. Cells were washed in phosphate-buffered saline (PBS) containing calcium and magnesium, fixed in 4% paraformaldehyde in PBS for 10 minutes, washed twice with PBS, and then permeabilized with 0.2% Triton-X100 in PBS for 5 minutes. Afterwards, cells were blocked in a PBS solution containing 5% bovine serum albumin (BSA) and 0.05% Triton-X100. Cells were first probed with a primary mouse monoclonal antibody against human HMOX1 (Abcam) diluted in PBS containing 1% BSA, 0.01% Triton X-100 for 1 hour, washed twice, and then probed with a secondary goat anti-mouse Alexa 488 antibody (Invitrogen) for 1 hour. Hoescht stain (Invitrogen) was included to identify cell nuclei. Stained cells were washed in PBS, and HMOX-1 was visualized using the InCell 2000 instrument (General Electric).

WM266-4 melanoma cells were pre labeled with 2μM CellTracker Orange CMTMR dye (ThermoFischer) as per manufacturer’s protocol. RNAi screens were performed in 96 well plates to which 160 nL/well siRNA (20 μM) were plated using an Echo liquid handler (LabCyte). Prior to seeding cells, 20 μL of OptiMEM (Invitrogen) containing 160 nL/well Lipofectamine RNAiMAX (Invitrogen) was added using a Multidrop Combi Reagent Dispenser (ThermoFischer) and plates were incubated for 30 min at room temperature (RT). One day after transfection cells were seeded on top of 100 μL of 1.8 mg/mL rat-tail collagen in DMEM plus 10% heat inactivated FBS prepared as per manufacturer protocol. Collagen was pre-aliquoted into wells of a 96-well, glass-bottomed, collagen-coated plates (PerkinElmer). After 24h of incubation cells were fixed by adding 100 μL of pre-warmed 8% methanol free formaldehyde (ThermoFischer) containing 5 μg/mL Hoescht stain (Invitrogen) and incubated for 1h at RT. Cells were imaged in the OPERA QEHS (PerkinElmer), with a 20x air immersion objective. Imaging was performed at a single timepoint capturing 15 wells per field and 200-plane z-stacks. Maximum intensity projections were used for image analysis.

High-content microscopy was carried out on an Array Scan XTI high content microscope (Thermo Scientific) using FITC-Tagged Annexin-V (BD-Biosciences) Propidium Iodide (Sigma-Aldrich), and Hoescht Stain (Invitrogen).

Following infection, cells were washed with 1× phosphate-buffered saline (PBS; Corning), fixed with 100 µL 4% paraformaldehyde (ThermoFisher, Waltham, MA, USA) for 20 min at room temperature and permeabilized for 15 min with 0.05% Triton X-100 (Sigma Aldrich, St. Louis, MO, USA). Viral capsid was detected by immunofluorescent microscopy as previously described [35 (link)]. Briefly, fixed monolayers were blocked for 1 h at room temperature with PBS containing 5% normal goat serum (NGS; Gibco). Cells were then incubated with anti-dsRNA (J2; Scicons, Szirák, Hungary), anti-HAstV-1 mouse monoclonal antibody 8e7 (Invitrogen ThermoFisher, Waltham, MA, USA) or anti-VA1 rabbit monoclonal antibody (generously provided by Dr. David Wang, Washington University School of Medicine in St. Louis, St. Louis, MO, USA) diluted in 1% NGS for 1 h at room temperature. Cell monolayers were then washed 3 times with 1× PBS and incubated with anti-mouse or anti-rabbit IgG labeled with either Alexa Fluor 488 or 555 (Invitrogen) secondary antibody and with Hoescht stain (ThermoFisher) at room temperature for 30 min. Imaging was performed on the EVOS FL cell imaging system followed by analysis with ImageJ 1.50i software.

iBMMs expressing ASC-citrine were plated on 96-well glass-bottom imaging plates, primed with 1 μg/mL of LPS for 3 h, then exposed to 39 °C for 1 h or left at 37 °C, and ATP (500 μM) was added for 30 min. Cells were then stained with MitoTracker Red CMXRos (Thermofisher, M7512) at a dilution of 1:5000 for 20 min and Hoescht stain (Thermofisher, H3570) at 1:10,000 for 10 min. Cells were washed in warm DMEM to remove excess stain. The cells were kept at 37 °C prior to imaging. Imaging was obtained using the ImageXpressMicro Confocal live cell imaging confocal system. ASC-citrine was obtained in the green channel, MitoTracker Red CMXRos in the red channel, and Hoescht stain in the blue channel. An algorithm was empirically designed by Dr. Lee Barrett using the custom module editor in MetaXpress 6 software to create masks defining threshold size and intensity for Hoescht-stained nuclei and MitoTracker-Red-stained mitochondria. In addition, numbers of ASC specks/DAPI nuclei were counted manually per 40× field, with greater than 5 fields analyzed per condition.