Cellmask red

The fluorescence immunostaining was conducted automatically using a microplate washer dispenser (EL406, BioTek Instruments, Winooski, VT, USA) under a programmed protocol. The plate was blocked with 1% bovine serum albumin in PBS (BSA/PBS) for 1 h at room temperature. After removal of the solution, 50 μL/well of anti-γH2AX (anti-γH2AX, phospho Ser139, mouse IgG [9F3], Abcam, Cambridge, UK) that had been diluted at 1:2000 with 1% BSA/PBS was added to each well and left for 2 h at room temperature. The plate was washed with PBS 3 times, and 50 μL/well of AlexaFluor488-labeled anti-mouse IgG goat antibody (Invitrogen) that had been diluted at 1:2000 with 1% BSA/PBS was added to each well, and then the plate was placed at room temperature for 2 h. After washing with PBS 3 times, the plate was fixed with 150 μL/well of 99.5% methanol for 5 min at room temperature. The methanol was removed and the air-dried plate was stored at − 30 °C until the time for staining. The plate was stained with 100 μL/well of PBS containing 1.4 μg/mL Hoechst33258 (Sigma) and 0.1 or 0.02 μg/mL CellMask Red (Invitrogen). The plate was stored at 4 °C until imaging.

Immunofluorescence staining was performed in four-chamber microplates (ThermoFisher Scientific) according to previously established protocols (25 (link), 28 (link), 33 (link)). The primary and secondary antibody set included unconjugated mouse anti-5-methylcytosine monoclonal antibody (AMM99021, Aviva Systems Biology) at 1 mg/ml and Alexa488-linked donkey anti-mouse IgG (A-21202, ThermoFisher Scientific) at 5 mg/ml final concentrations. The cells were subsequently delineated with the cytoplasmic marker Cell Mask Red (ThermoFischer Scientific) and cell nuclei counterstained with DAPI. The specificity/dynamic range of the anti-5mC antibody was tested as previously reported in (29 (link)) (data not shown in here). Formalin-fixed tissue sections at 5 µm thickness were kept floating in 10% formalin at 2−8°C until immunofluorescence staining. Prior to staining, tissues were transferred to microwell plates, washed in PBS at room temperature, then stained as floating tissues using the same protocol that was applied to fixed cells.

HUVEC were plated at 10 K cells/well in 96 well plates (Imaging plates; Falcon black-walled #353219) and were later IFNγ stimulated after cells had attached. After 18 h, cells were infected with type II Toxoplasma-GFP at MOI = 1–2 for 6 h. Plates were washed in PBS to remove uninvaded parasites and fixed in 4% PFA 15 min. The cells were permeabilized with PB (0.2% BSA, 0.02% Saponin in PBS) for 30 min before staining with Hoechst 33342 5 ug/mL and Cell Mask Red (ThermoFisher #H32712). After washing twice with PBS, the stained plates were imaged on Zeiss Cell Discoverer 7 using 20× objective and 0.5× magnification. Images were analyzed using Zen Blue software, and infection analysis performed by host response to microbe analysis pipeline to obtain values for percentage infection and vacuole/cell ratio. For recruitment analysis, cells were plated and infected for 2 h before fixation as above and were then permeabilized and stained with antibody, according to the immunofluorescence protocol below. Recruitment was counted manually (>200 vacuoles per condition) since the HRMAn pipeline had not been trained for these cells and stains.

PC3 cells were stained with Mitotracker Green FM (mitochondria, ThermoFisher, Waltham, MA, USA, Cat#M17514), cell mask red (cell membrane, Thermo Fisher Cat#A57245), and hoechst (nucleus, Thermo Fisher Cat#H3570). Images were then taken with the Nikon A1 confocal microscope using a Z-stack (Z range:10.00 µM, Step:1.00 µM). The images from the NIS-Element (v5; Nikon Instruments Inc. Melville, NY, USA) were converted into Imaris files (IMS format) by Imaris rendering software (v9.6; Oxford Instruments, Abingdon, UK) for 3D visualization and DATA processing. Border of each cell was traced based on cell mark fluorescence intensity. Green fluorescent intensity represents mitochondrial mass in each cell was then segmented and quantified based on automated relative fluorescence algorithm provided by Imaris software. The following parameters were collected: Green intensity mean per mitochondrion, volume per mitochondrion based on GFP, Sphericity of mitochondrion, and Ellipticity of mitochondrion.

The immunofluorescence staining of zonula occludens-1 (ZO-1, also called tight junction protein-1) was performed as follows. Cells, grown on polylysine-treated coverslips, were washed in PBS, fixed in ice-cold methanol for 7 min at −20 °C, permeabilized with 1% triton X-100 for 5 min at room temperature. Non-specific binding sites were blocked with blocking buffer (3% BSA in PBS) for 1 h at room temperature. Cells were labelled with anti-ZO-1 primary monoclonal antibody (1A12) (Invitrogen) (1:50) overnight at 4 °C in a humidified atmosphere follower by alexafluor-488 anti-mouse secondary antibody (Invitrogen) for 1 h at room temperature. After washings in PBS added with1% tween-20, nuclei were counterstained with 4,6′-diamidino-2-phenylindole (DAPI).
Morphological changes of plasma membranes were analyzed in live cells, grown on polylysine-treated coverslips, were incubated for 10 min with CellMask red (Thermo Fisher Scientific) in cell culture medium following manufacturer’s instruction. After washings in PBS, nuclei were counterstained with DAPI.
Cells were then rinsed in PBS, mounted and analysed with a Zeiss Axio Observer Z1 equipped with Apotome and digital Camera Axiocam MRm (Zeiss, Oberkochen, Germany).

ZsG fluorescence in infected cells was imaged directly using an EVOS digital inverted microscope (Thermo-Fisher, Waltham, MA, USA). For immunofluorescence, cells were fixed in 10% formalin for 30 min, permeabilized in PBS containing 0.1% Triton-X100 (v/v) for 10 min, and blocked with PBS 5% BSA (v/v) for 30 min. Primary antibody was rabbit α-SOSV NP (Genscript, Waltham, MA, USA), visualized with an AlexaFluor 488-labeled secondary antibody (Thermo-Fisher, #A11034). Quantification of total rSOSV protein production in Huh7 cells was performed on infected cells labeled as described above, and further stained with Nuc-Blue and Cell-Mask Red (Thermo-Fisher). Images were collected on the Operetta high-content imaging system (PerkinElmer, Waltham, MA, USA) using a 20× objective, with 16 fields per well analyzed using the Harmony software package (PerkinElmer). Total 488 fluorescence per field was determined, averaged for the well, and then normalized to values from control wells containing rSOSV-infected, mock-treated cells.