In cell analyzer 2000 system

HEY and A2780 cells were seeded into 96-well plates at a cell density of 4 × 10³ cells per well, cultured overnight, and treated with GE diluted in the medium containing 0.5% FBS for 24 h. The suspension was removed, and the cells were fixed with 4% PFA (Sigma) for 20 min. 1 μg/mL of Hoechst 33342 (Sigma) was added and incubated for 15 min in the dark. Apoptotic morphological changes in the nucleus were observed using an In Cell Analyzer 2000 System (GE Healthcare, Uppsala, Uppsala, Sweden). The experiments in this assay were performed three times.

To detect protein expression levels in both undifferentiated and differentiated cells, the monoclonal antibody TRA-1-60 (Santa Cruz Biotechnology) was used as the primary antibody to assess surface antigens on the NT2 cells and the changes that occur upon differentiation induced by RS1S CHCL₃ extract. Following the treatment, cells were washed and then fixed in 4% paraformaldehyde (20 min) at room temperature. Fixed cells were incubated with blocking buffer (3% FBS in 1× Phosphate buffer saline (PBS)) for 40 min, and then the cells were incubated overnight at 4 °C with TRA-1-60 primary antibody diluted 1:100 in blocking buffer. After incubation, the cells were washed 3 times with 1× PBS and then incubated for 1 h at room temperature on a shaker with FITC-conjugated goat-anti-mouse IgM (Santa Cruz Biotechnology) secondary antibody diluted 1:500 in blocking buffer, and the cells were then washed 2 times with 1× PBS. For nuclei staining, the cells were incubated with 1× PBS containing 0.5 μg/ml Hoechst (Sigma) for 5 min. The stained cells were imaged using an In Cell Analyzer 2000 System (GE Healthcare Life Sciences, USA). For the negative control, conditions were kept the same, except that the primary antibody was omitted.

Cells were seeded on 96-well plates, and mature junctions were allowed to establish over 2 days. When the experiment was done, cells were washed once for 1 min with PBS and fixed for 20 min with 4% formaldehyde in PBS. After being rinsed once with PBS, cells were permeabilized with PBS containing 0.3% Triton X-100 for 20 min on ice. After being washed three times with PBS for 1 min, cells were blocked with 2% BSA in PBS for 1 h at room temperature. Primary antibodies were applied in 2% BSA in PBS and incubated overnight at 4 °C. Cells were then washed three times for 1 min in PBS. Secondary antibodies conjugated to fluorescent probes (Alexa Fluor 488 rabbit anti-goat IgG (H+L), Molecular Probes, Thermo Fisher Scientific Inc.) were applied for 1 h at room temperature. Cells were washed 4× with PBS for 5 min per wash and images were taken with the IN Cell Analyzer 2000 system (General Electric, Marlborough, MA, USA).

The invasion ability of cells was examined by using HTS FluroBlok^TM Insert containing a 6.5-mm filter with a pore size of 8 µm (BD Falcon, San Jose, CA, USA). For Matrigel coating, 50 µl of 100 µg/mL Matrigel (BD Biosciences, Bedford, MA, USA) was applied to the membrane filters of the insert. Filters were dried overnight in a laminar flow hood. Colon-26MGS cells and Colon-26 cells were pre-labeled with DiIC12(3) (BD Biosciences) at a concentration of 1.25 µg/mL for 1 h at 37 °C, 5% CO₂. Pre-labeled cells were suspended into the serum-free RPMI1640 medium and seeded into each of the upper inserts at 2 × 10⁴ cells/well, while RPMI-1640 supplemented with 10% fetal bovine serum was added in the lower chamber, and the real-time kinetic invasion assays were carried out for 36 h. The images of total cells and invaded cells were captured at different time points using the In Cell Analyzer 2000 system (GE Healthcare, Buckinghamshire, UK). The number of invaded cells in three random fields was counted using ImageJ software. Each experiment was performed in triplicate (n = 3).

Vesicular stomatitis virus (VSV) (Indiana) containing the green fluorescent protein (GFP) gene instead of the VSV glycoprotein (G) gene (VSVΔG*-G) and pseudotyped VSVs bearing GPs of LASV (strain Josiah) or LUJV (strain IGR140) were generated as described previously (15 (link), 34 (link)). Briefly, HEK293T cells were transfected with the expression plasmid pCAGGS carrying the LUJV GP gene, and 24 h later, the cells were infected with VSVΔG*-G at a multiplicity of infection of 1.0. After a 16-h incubation, the supernatants were collected and centrifuged to remove cell debris. Infectious units (IU) of the pseudotyped VSVs in each cell line were determined as described previously (35 (link), 36 (link)). Briefly, cells were seeded into 96-well plates 1 day before virus inoculation, and 24 h later, GFP-positive cells were counted using an In Cell analyzer 2000 system (GE Healthcare). To reduce the background infectivity of the residual parent VSVΔG*-G, each pseudotyped virus stock was treated with a neutralizing monoclonal antibody specific for the VSV G protein (VSV-G[N]1-9) before use (37 (link)).