Alexa fluor 488 conjugated anti mouse antibody

This assay was performed similarly to one previously described (29 (link)). Briefly, LF-6 (20 μM) was premixed with soluble F(ecto), F(ecto)_K394R, or F(ecto)_ΔFP at 4°C for 20 min. HEp-2 cells were incubated with the mixtures of LF-6 and soluble F proteins for 1 h at 37°C. Afterward, the cells were incubated with anti-RSV monoclonal antibody (catalog no. ab94968; Abcam) in PBS for 1 h, followed by staining with Alexa Fluor 488-conjugated anti-mouse antibody (Thermo Fisher Scientific) at 37°C for 1 h. Mean fluorescence intensity (MFI) correlated with the amount of F protein on the cell surface was measured by flow cytometry (BD Biosciences). All of the soluble proteins were used at a final concentration of 2 μg/ml. Data were analyzed using FlowJo v.10.

Indicated MCF-7 and T-47D cells (1 × 10⁴) were plated on coverslips and after overnight culture, cells were treated with 1 µM OTX015 or DMSO for 24 hours followed by IR (4 Gy). 30 minutes and 120 minutes after IR treatment, cells were fixed in 4% paraformaldehyde, permeabilized using PBS with 0.3% Triton-X, and blocked with 5% goat normal serum. The coverslips were incubated overnight with Ser139 phosphorylated histone H2AX (γH2AX) antibody (1:1000 dilution; Cat #05-636, Millipore), followed by Alexa Fluor 488 conjugated anti-mouse antibody (1:200 dilution; Cat# A-21121, Thermo Fisher Scientific). The nuclei were counterstained and coverslips were mounted using ProLong Gold Antifade Mounting media with DAPI (Thermo Fisher Scientific). Images were captured by fluorescence microscopy (Zeiss, #LSM 880) and foci in the nucleus (at least in 40 nuclei for each treatment group of the experiment) were counted manually and plotted as the number of foci per nucleus.

One day prior to infection, 8-well chamber slides (Lab-Tek) were seeded with 4 10⁵ Huh-7.5 cells/well. Infections were performed with 10-fold serial dilutions of viral samples in 100 μl for 4 h, after which the supernatant was replaced with fresh media. Three days post-infection, slides were fixed in 100% acetone and stained with anti-HCV core antibody (1:100, clone B2, Anogen), and subsequently with the AlexaFluor-488-conjugated anti-mouse antibody (1:200, ThermoFisher Scientific) for immunofluorescence analysis. Viral titers are expressed as the number of focus-forming units (FFU) per ml. Extracellular virus titers were determined directly from cell supernatants.

293T cells transfected with FLAG-tagged vector, ASXL1 (MT or MT-K351R) and HA-tagged BAP1 (WT or C91S) were fixed with 2% paraformaldehyde, permealized with 0.2% Triton-X, blocked with 2% BSA and 5% goat serum, and were then incubated with anti-FLAG rabbit monoclonal antibody (Sigma-Aldrich, catalog #F7425, 1:200) and anti-HA mouse monoclonal antibody (BioLegend, catalog #901513, 1:200), followed by labeling with Alexa Fluor 568-conjugated anti-rabbit (Thermo Fisher, catalog #A11011, 1:1000) and Alexa Fluor 488-conjugated anti-mouse antibody (Thermo Fisher, catalog #A11029, 1:1000). Nuclei were visualized with DAPI (BioLegend catalog #422801, 1 μg/ml). Fluorescent images were analyzed on a confocal microscope (Nikon A1).

Cells were seeded on coverslips in a 12-well plate and incubated overnight in RPMI 1640 supplemented with 10% FBS and 1% P/S. Cells plated on coverslips were incubated with 4% paraformaldehyde for 10 mins at room temperature and washed three times with 1× PBS. Cells were then incubated with 0.5% Triton X-100 for 2 mins at room temperature and washed three times with 1× PBS. Coverslips were incubated with blocking buffer for 30 mins at room temperature. Coverslips were then incubated with mouse anti-vinculin antibody (Sigma-Aldrich, V4505, batch #0000216740, 1:500) overnight at 4°C. Coverslips were then washed three times with 1× PBS and incubated with Alexa Fluor 488-conjugated anti-mouse antibody (Thermo Fisher Scientific, A21202, 1:500) and Alexa Fluor 647-conjugated Phalloidin (Thermo Fisher Scientific, A22287, 1:500) for 30 mins at room temperature. After three washes in 1× PBS, cells were incubated with DAPI (Cell Signaling Technology, 4083S, 300 nM) for 5 mins. Coverslips were then washed twice with 1× PBS and once with distilled water before mounting the coverslips onto microscopy slides using 4 µl Mowiol. Coverslips were imaged on a Zeiss Axioplan2 microscope with a 63×/1.4 NA Plan-Achromat objective and a Photometrics Cool Snap HQ cooled charge-coupled device camera. Data for 20-30 cells per sample were analyzed using FIJI.

HEp-2 cells grown in flasks were dissociated and then centrifuged for 5 min at 4°C and 1,500 × g. The pelleted cells were resuspended in 4°C precooled medium containing the mixtures of virus suspension with LF-6 or heparin at different concentrations. Afterward, the cells were gently rocked for 1 h at 4°C to allow viral attachment to the cell membrane. Following the incubation, the unbound virus on the cell surface was removed by centrifugation at 2,000 × g at 4°C for 3 min and then washed twice with PBS. Afterward, cells were fixed with 4% paraformaldehyde for 20 min at 4°C. Following the washing twice with PBS, cells were incubated with mouse anti-RSV F monoclonal antibody (catalog no. ab94968; Abcam) for 2 h at RT and followed by staining with Alexa Fluor 488-conjugated anti-mouse antibody (Thermo Fisher Scientific) at RT for 1 h. Finally, cells were washed twice with PBS and subjected to flow cytometry (BD Biosciences, CA, USA). Data were analyzed using FlowJo v.7.6.

One day prior to infection, 8-well chamber slides (Lab-Tek, Brendale, Australia) were seeded with 4 × 10⁵ Huh-7.5 cells/well. Infections were performed with 10-fold serial dilutions of viral samples in 100 µL for 4 h, after which the supernatant was replaced with fresh media. Three days post-infection, slides were fixed in 100% acetone and stained with anti-HCV core antibody (1:100, clone B2, Anogen, Mississauga, ON, Canada), and subsequently with the AlexaFluor-488-conjugated anti-mouse antibody (1:200, ThermoFisher Scientific, Waltham, MA, USA) for immunofluorescence analysis. Viral titers are expressed as the number of focus-forming units (FFU) per mL.
Extracellular virus titers were determined directly from cell supernatants, while intracellular virus titers were determined after cell pellets were subjected to lysis via four freeze-thaw cycles, removal of cellular debris via centrifugation, and recovery of virus-containing supernatants.