Xcelligence sp system

Cells were seeded at 1500 cells per well in 200 μl of complete media as described above in E-plates (ACEA Biosciences) and grown overnight while monitored with an xCELLigence SP system (ACEA Biosciences) which monitors cellular events in real time by measuring electrical impedance across interdigitated gold micro-electrodes integrated on the bottom of tissue culture plates [56 (link)]. Cells were washed three times with PBS and replaced with 180 μl of low nutrient media as described above and incubated for a minimum of 6 h before further treatments. Treatments were prepared at 10× concentrations and added to each well in a total volume of 20 μl. The xCELLigence system recorded cell index readings hourly for 5–6 days after treatment. Cell index readings were normalized before treatment and cell proliferation ratios were determined from biological triplicates and represent the relative numbers of cells compared to control cells.

The human skin normal fibroblast cell line, 1BR.3.GN, obtained from the European Collection of Authenticated Cell Cultures was confirmed to be mycoplasma free and cultured to test effects of TxVIIB on cell proliferation on a xCELLigence SP system (ACEA Bioscience) as described previously (18 (link)). A plant extract, which previously shown enhanced cell proliferation (unpublished results), was used as a positive control in the assay, serum-free media was used as a negative control. Day 4 cell proliferation rates were compared between treatment and control wells, and a one-way ANOVA test was used for multiple comparisons with Holm-Sidak’s correction, using GraphPad Prism 9.3.1.

Cells were seeded at 5000 cells/well in 170 μL of complete media in E-plates (ACEA Biosciences, San Diego, CA, United States) and grown overnight and monitored with an xCELLigence SP system (ACEA Biosciences), which monitors cellular events in real time by measuring electrical impedance across gold microelectrodes integrated into the base of tissue culture plates. Cells were washed three times with PBS prior to addition of 150 μL of low nutrient media and incubated for a minimum of 6 h before further treatment. Treatments were prepared at 8.5 × concentration and added to each well in a total volume of 20 μL. The xCELLigence system recorded cell indexes at intervals of 1 h for 5−6 days following treatment. Readings for the cell index were normalized prior to treatment, and cell proliferation ratios represent the relative numbers of cells compared to control cells at day 4. Comparisons of induction of cell proliferation in response to treatments were accomplished using the Two-Way ANOVA test with Dunnett’s multiple comparison correction, using GraphPad Prism 8.0.

5 × 10³ cells were seeded in a 16-well E-plate (Roche) and cultured in a 5% CO₂ incubator at 37°C. Cell growth was continuously monitored for 72 h by xCELLigence SP system, which used the Real Time Cellular Analysis (RTCA) technology developed by ACEA Biosciences, Inc. (San Diego, CA, USA). The xCELLigence^® RTCA DP instrument (Roche) was used to dynamically monitor proliferation and cell viability in a label-free manner. Data were tabulated and analyzed using the specialized RTCA software.

The xWORM technique was employed using an xCELLigence SP system (ACEA Biosciences Inc., USA) as described previously16 45 (link). All experiments were carried out as per the manufacturer’s instructions with 15 sec read intervals using the real time cell assay (RTCA) software (ACEA Biosciences Inc., USA). Separated (single) and paired adult S. mansoni flukes per well (mixed gender) were used in the xWORM assay (two independent experiments), and four adult T. muris of mixed gender were placed in each well of the 96 well E-plates containing a final volume of 200 μl of culture medium. All assays were conducted in triplicate. Inter-well spaces were filled with 100 μL of culture medium or PBS to prevent evaporation. The E-plates containing worms were cultured overnight at 37 °C with 5% CO₂ to obtain a baseline motility reading. The worms were then treated with prepared concentrations of the test compounds and the motility of the worms was monitored for 12–40 h.

Full-length Hsp20 was cloned into pcDNA3.1/V5-His-TOPO vector (Invitrogen) and related mutants created using Quikchange (Stratech). The various Hsp20 constructs and an empty vector control were electroporated into SH-SY5Y cells using nucleofection kit V (Amaxa) in accordance with manufacturer's instructions. Cell were seeded at a density of 5 × 10³/well into seeded into 96-well plate for MTT-based assays or 96-well E-plate for xCELLigence based assays and left overnight to allow for cell re-attachment. Remaining cells were seeded into 6 well plates and harvested after 48 h to confirm expression of the various Hsp20 constructs. Addition of Aβ peptides and vehicle controls was carried out once cell index reached 1. The xCELLigence SP system (Acea) was used for real-time monitoring of cell growth for a minimum of 48 h post addition of Aβ peptides. The resulting data was analysed using (RTCA) real-time cell analyzer software (Roche) and exported to Excel. MTT based cell viability was carried out in parallel in accordance with Promega CellTiter 96® non-radioactive cell proliferation assay (G4000) in accordance with manufacturer's protocols and added 48 h post addition of Aβ peptides.