E plate 96

Exosome collection: Equivalent numbers of KYSE150-pcDNA3.1 and KYSE150-PCAT1 cells were plated in exosome-free medium and cultured for 48 h. The supernatant from cells was collected and then centrifuged at 3000 rpm for 20 min to remove cell debris. The supernatant was concentrated to 1 mL using a Millipore ultrafiltration centrifuge tube (Millipore, USA).
Exosome isolation: Exosomes in supernatant were isolated with the Ribo^TM Exosome Isolation Reagent (RiboBio, China) according to the following protocols: The concentrated supernatant was transferred to a new tube, and 1/3 volume of Ribo^TM Exosome Isolation Reagent was added. Each sample was mixed well by inverting or flicking the tube, and it was incubated at 4 °C overnight, after which the solution appeared cloudy. Each sample was centrifuged at 15,000 × g for 2 min at 4 °C. We carefully aspirated off the supernatant, and resuspended the pellet in 400 μL of PBS, and stored it at 4 °C.
Exosome treatment: NE3 cells were seeded at 2 × 10³ cells/well into an E-Plate 96 (Roche Applied Science), and 5 μL of KYSE150-pcDNA3.1 and KYSE150-pcDNA-PCAT1-derived exosomes were added at 0, 24, 48 and 72 h. Cell growth was monitored by the RTCA-MP system at 37 °C with 5% CO₂.

The proliferation ability of glioma cells was monitored by using the xCELLigence Real-Time Cell Analyzer (RTCA)-MP system (Acea Biosciences/Roche Applied Science). This platform is able to measure cellular growth status in real time. First, 100 μl of culture medium was added to each well of E-Plate 96 (Roche Applied Science) to obtain equilibrium. Then, 2,000 cells in 100 μl of culture medium were seeded in each well. E-Plate 96 was locked in the RTCA-MP device at 37 °C with 5% CO₂. Measured changes in electrical impedance were presented as a cell index that directly reflects cellular proliferation on biocompatible microelectrode coated surfaces. The cell index was read automatically every 15 min, and the recorded curve was depicted as the cell index ± SD.

The growth ability of ESCC cells was evaluated using the xCELLigence Real-Time Cell Analyzer (RTCA)-MP system (Acea Biosciences/Roche Applied Science), which can detect the cellular growth status in real time. First, 100 μL of RPMI 1640 complete medium was added to each well of an E-Plate 96 (Roche Applied Science) to obtain equilibrium. Then, 2 × 10³ cells in 100 μL of RPMI 1640 complete medium were seeded in each well. The E-Plate 96 was locked in the RTCA-MP device and continually cultured at 37 °C with 5% CO₂. The cell index obtained from changes in electrical impedance directly reflects cellular proliferation on biocompatible microelectrode-coated surfaces and was read automatically every 15 min until the end of the experiment.
For the drug-sensitive assay, briefly, 5 × 10³ cells per well were plated in 96-well plates. After overnight culture, the cells were treated with a series of concentrations of paclitaxel. 48 h later, 10 μL of MTT solution was added to each well, followed by incubation for 2 h and subsequent addition of DMSO. The plate was then shaken and measured with a microplate reader (Thermo Scientific, USA) using a 492 nm filter.

This assay is a real-time technique to dynamically monitor cell viability. Microelectrode-coupled microtiter plates were used to culture cells, and the cell number, cell morphology, and degree of cell adhesion were reflected by the electrode impedance. Untreated cells attach to the bottom of the well and cause an increase of the cell index (CI) value due to the increased impedance. When treated with toxic compounds, the cells die and detach from the bottom, displaying a decrease of the CI value. 50 μl of culture medium was added to each well of the E-plate 96 (Roche) for background measurements. NIH 3T3 cells were then added at a concentration of 5000 cells per well for 24 hr incubation, followed by adding PG-M and morphine sulphate diluted with DMEM to contain morphine-free base concentrations of 2.6 to 168 μg/ml. Untreated cells were used as controls. The E-plate was incubated and monitored on the RTCA SP system (Roche) for 48 hr at intervals of 15 min for 48 hr. The normalized CI was calculated by CI _original/CI _{normalize time} using the RTCA software version 1.2.1. Four replicates were measured for each sample and the means are reported.