Axioobserver z1 inverted fluorescent microscope

Cells were seeded on coverslips in a 24-well plate at a density of 100x10³cells/mm². After an overnight starvation, cell death was examined by TUNEL assay using the In Situ Cell Death kit (Roche, Basel, Switzerland) as per manufacturer’s instructions. To visualize the nuclei, cells were incubated with DAPI for 5 minutes. Samples from three independent experiments were evaluated by Zeiss Axio Observer Z1 Inverted fluorescent microscope (USA). 200-300 cells were counted for each image taken from at least 6 independent areas per sample.

To analyze cellular binding and uptake of Pd[DMBil1]-PEG₇₅₀ and PEG-NSs by fluorescence or darkfield imaging, cells were plated at 15,000 or 25,000 cells/well, respectively, in glass bottom 8-well plates with removable well chambers, and were incubated overnight. Then, 2.7 × 10⁹ PEG-NSs/mL and/or 1 mM Pd[DMBil1]-PEG₇₅₀ diluted in complete cell culture medium was added to cells for 24 h protected from light. Cells were fixed with 4% formaldehyde for 15 min and rinsed three times with 1X PBS. Cells were then stained with phalloidan (Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C to visualize F-actin on the cell cytoskeleton. Well chambers were removed and slides were mounted with DAPI-containing mounting media to label cell nuclei (Vectashield, Burlingame, CA, USA). Cells were imaged with a Zeiss Axioobserver Z1 Inverted Fluorescent Microscope (Oberkochen, Germany) using the FITC (F-actin), DsRed (Pd[DMBil1]-PEG₇₅₀), and DAPI (nuclei) fluorescence channels and a darkfield condenser (to visualize scattering from PEG-NSs).

Proliferation was evaluated using In Situ Cell Proliferation assay kit (Roche, Basel, Switzerland) per the manufacturer’s instructions. Briefly, cells were seeded on 24-well plates at a density of 100x10³cells/mm², and subsequent to serum starvation overnight they were induced with 1 ng/ml FGF2 (R&D Minneapolis, USA) for 24 hour. After extensive washing steps with PBS, the cells were incubated in a medium containing 10 μM BrdU for 5 hours. After removal of BrdU labelling solution, cells were incubated in blocking solution for 1 hour and treated with anti BrdU Fluos antibody. Cell nuclei were stained with DAPI. Samples from three independent experiments were examined using Zeiss Axio Observer Z1 Inverted fluorescent microscope (USA). 200-300 cells were counted for each image taken from at least 6 independent areas per sample.

Huh 7.5.1 cells were seeded at 100,000/slide in a 24-well plate and cultured in DMEM+ 10% FBS for 24 h. They were subsequently infected with SARS CoV-2 at MOI of 2 for 1 h in a 5% carbon dioxide incubator at 37 °C. The inoculum removed and replaced with complete media containing 1% DMSO or 10 μM GSK3β inhibitor. Uninfected cells received only complete media. Cells were left to grow for another 2 days, washed with cold PBS and fixed with 4% paraformaldehyde for 30 mins. The fixative was removed, cells washed with PBS, and permeabilized with 0.1% Triton × 100 for 4 min. Cells were again washed with PBS and blocked with 1% BSA for 1 h. BSA was removed and cells were incubated with the primary antibodies mouse monoclonal antibody to S1 protein (GTX635708, 1:500) and rabbit polyclonal antibody to nucleocapsid protein (GTX635679, 1:1000) for 1 h at room temperature. Secondary antibodies were used at a 1:1000 dilution, incubated for 1 h with Fluoroshield DAPI (Abcam, Ab 104,139) for nuclear staining, removed, washed with PBS, and mounted in aqueous mounting medium. Images were captured on the Zeiss Axio Observer Z1 inverted fluorescent microscope (Zeiss, Germany). The fluorescent intensity was calculated using ImageJ software.

Cohesin‐dockerin binding was measured by ELISA (Barak et al., 2005). The interaction between the GFP‐tagged Amy4 dockerin (GFP‐doc‐13a) protein and R. bromii cells was assessed using 24 h cultures of R. bromii L2–63 grown in M2S medium (OD₆₅₀=0.6). After centrifugation the cell bacterial pellet was washed with sterile PBS and divided into three separate tubes. In the first tube, the pellet was re‐suspended in PBS, treated with 10 mM EDTA and washed with PBS three times. Cells were then treated with GFP‐doc‐13a protein (40 μl) along with 10mM calcium and incubated for 1 h at room temperature on a rotating platform. The cells were then washed with PBS three times to eliminate any unbound protein, centrifuged at 5000 rpm for 10 min, re‐suspended in PBS, smeared on microscope slides, air‐dried, overlaid with Vectashield anti‐fade mounting Medium (Vector Laboratories, Peterborough, UK) and examined with a Zeiss Axio Observer Z1 inverted fluorescent microscope (Carl Zeiss, Cambridge, UK) using a Zeiss 10 FITC filter. The R. bromii pellet from the second tube was treated exactly as the first tube but without the addition of calcium. The third tube was a control where the bacterial pellet was simply washed in PBS.

Confluent OECs grown on coverslips were treated with rSap6, rSap6 (Inact), PAR2-AP and PAR2-ANT for 2 or 24 h. After treatment, cells were then fixed with 4% para-formaldehyde in PBS for 20 min at 20°C and washed three times for 5 min with PBS. Fixed cells were blocked with 1% BSA for 1 h, then incubated with primary antibodies for Par2 (Cell Signaling) or E-cadherin (Santa Cruz) at 1:1000 for 12 h at 4°C. Cells were again washed three times with PBS before addition of the secondary antibody (anti-rabbit IgG Alexa Fluor^® 488; Abcam) for 2 h at room temperature. Cells were stained with Hoechst (Sigma) to stain nuclei, washed, and cover glass slips were mounted on slides (Globe Scientific Inc.) using fluorescent mounting media (Dako). Cells were imaged using a Zeiss Axio Observer Z1 inverted fluorescent microscope (Carl Zeiss, Germany). The images were processed using ImageJ software.