Xcelligence

Biofilm formation was measured using the real time cell analyzer xCELLigence (Agilent Technologies, USA), as described previously for Streptococcus mutans [21 (link)]. This equipment allows for the detection of variations in the impedance signal, due to bacterial attachment and biofilm formation on the gold-microelectrodes present at the bottom of the E-plates (Agilent Technologies, USA). An initial baseline impedance reading of 150 µL of blank media for the sample wells, or 200 µL of media for the controls wells, was taken prior to bacterial cultures being added to wells. Bacteria were initially grown overnight on Columbia blood agar. Bacterial cells were resuspended in CDM+Glc to OD₆₀₀ of 0.2. Fifty µL of bacterial cell suspension was added to the wells of the E-plate for a ¼ dilution. The xCELLigence system harbouring the bacterial cell cultures was incubated at 37 °C for the duration of the experiment. Biofilm formation was monitored over 24 h by recording the impedance signal at 15 min intervals.

Growth rates were determined by real-time cell analysis (RTCA, xCELLigence; Agilent, Santa Clara, CA, USA). A number of 10,000 cells were seeded per well in special 96-well E-plates equipped with electrodes in the bottom of wells, and treatments started after 24 h. Electric resistance was continuously determined up to 140 h, which served as a measurement of cell confluence. A second cell proliferation assay was based on WST-1 staining (Water soluble tetrazolium-1, Roche Diagnostics), further analyzed by ELISA.

Tumor cell migration and monocyte invasion were monitored using xCELLigence E- and CIM-Plates (Agilent, Santa Clara, CA, USA) according to the manufacturer’s protocols. Migration assay: A CIM-Plate was used to monitor cell migration across a porous membrane (xCELLigence, Agilent, Santa Clara, CA, USA) using RTCA [27 (link)]. HUVEC were plated in the bottom well and treated with 2 µM palbociclib for four hours. MDA-MB-231 cells were then added to the top chamber, which contained a porous membrane and electrode to detect migrating cells. Changes in the electric impedance as cells migrated between chambers was measured at 5 min intervals. Invasion assay: HUVEC (30,000 cells per well) were plated on an E-plate for 24 h until a monolayer was formed. The palbociclib monolayer was then treated with 5 ng/mL recombinant interleukin-1-β (Peprotech, Waltham, MA, USA) and/or varying concentrations of palbociclib for 4 h. THP-1 cells (10,000 per well) were then added to the monolayer to determine their invasive capacity. Graphs show representative data from one of three independent experiments.

Invasive potential was assessed using a real-time cell analyzer (xCELLigence, Agilent, Santa Clara, CA, USA) along with NCC-MFS1-C1 [28 (link)], NCC-MFS2-C1 [29 (link)], NCC-MFS3-C1 [30 (link)], and NCC-MFS4-C1 [31 (link)], as previously reported [32 (link)]. We seeded cells (4 × 10⁴) suspended in a serum-free DMEM/F12 medium in the upper chamber coated with BD Matrigel matrix (BD Biosciences, Franklin Lakes, NJ, USA) (n = 2). DMEM/F12 supplemented with 10% FBS (Gibco), 100 μg/mL penicillin, and 100 μg/mL streptomycin (Nacalai Tesque) were added to the lower chamber. The migration of cells from the upper to the lower chamber was measured by electronic sensors every 15 min for 72 h.

To compare growth rates of MIN6 cells under influence of the wild type and mutant INS cDNA expressing constructs, 2 × 10⁶ cells were seeded into 6-well plates and transfected with GFP tagged wild type and mutant constructs, according to the manufacturer’s recommendations. pMAX-GFP (Lonza, Basel, Switzerland) was used as a control. Transfection efficiency was routinely 40%. After 2 days, transfection cells were sorted using Sony MA900 sorter (Sony Biotechnology Inc.) and plated for additional 4 days to grow. For proliferation measurement, cells were seeded in two replicates into wells of a 16-well xCELLigence (Agilent, Santa Clara, CA, USA) plate, according to the manufacturer’s recommendations. As the xCELLigence in real time measures the net adhesion of cells to high-density gold electrodes, we analyzed cell index curves. As a baseline background, we used the cell index level from the culture medium with no cells. We measured the cell growth before a plateau phase of proliferation. The curves represent the mean cell index value from minimum 2 wells ± SD. To compare the growth rate, the slope of the linear region of the cell index curve was calculated using the internal xCELLigence software. Transfection, sorting, and proliferation measurements were repeated twice.

Invasion assays were performed using a real-time cell analyzer (xCELLigence, Agilent, Santa Clara, CA, USA). Briefly, the cells (4 × 10⁴) suspended in serum-free DMEM/F12 medium were seeded in the upper chamber coated with BD Matrigel matrix (BD Biosciences, Franklin Lakes, NJ, USA). The culture medium, which was used to maintain the cells, was added to the lower chamber. Then, the electronic sensors detected the cells that migrated from the upper chamber to the lower chamber. Measurements were taken every 15 min for 72 h. In this experiment, MG63 osteosarcoma cells (JCRB; Ibaraki, Osaka, Japan) were used as controls [34 (link)].

Using a label-free cellular impedance method (xCELLigence; Agilent, RTCA software 2.1.0) we continuously monitored cytopathic effect indicating non-neutralized virus growth in the presence of saturating neutralizing concentrations of mAbs RVFV-140, RVFV-268, or the combination of the two mAbs^{75 (link)–78 (link)}. Antibody solutions at 1 µg/mL total concentration were mixed 1:1 in 5% FBS-supplemented DMEM with RVFV strain MP-12 (~35,000 PFU per well) and incubated for 1 h at 37 °C and then added to cells. Two controls were included, a virus-only (no-mAb) and medium-only (no-virus) mixture. Selection and amplification of virus mutants that were not neutralized by the antibodies is detected when a drop in cellular impedance occurs in a well during a 96-h incubation with continuous real-time measurements. Since we did not observe any well to exhibit a drop in impedance, we were unable to identify a mutant virus using this methodology with RVFV strain MP-12 for mAb RVFV-140, RVFV-268, or the combination of the two mAbs.