Propidium iodide

Spheroid cultures were measured using self-developed microcavity arrays with pyramidal cavities (edge length 200 µm, depth 100 µm) in combination with a self-developed multiplexer system^{24 (link)} and the high precision impedance analyzer ISX-3 (Sciospec Scientific Instruments, Germany). Impedance spectra were recorded from 5 kHz to 5 MHz (51 points, 100 mV amplitude). For cell migration on high-dense microelectrode arrays, the array surface was coated with collagen I (Life Technologies, Germany) for 1 hour. Afterwards, individual spheroids were placed centrally in each well. Impedance spectra were recorded for 144 hours from 5 kHz to 5 MHz (41 points, 10 mV amplitude) using an extended multiplexer system with 378 channels and the high precision impedance analyzer ISX-3. Raw data were analyzed and processed with the self-developed software IDAT v3.6. The relative impedance (extracted cell signal) was calculated as follows: (IZI with cells - IZI without cells) / ZI without cells × 100%. Finally, vital staining was performed by 10 min incubation with 2.5 µM calcein AM (eBioscience, Germany), 1 µM propidium iodide (VWR, Germany) and 1 µg/mL Hoechst 33342 (Life Technologies, Germany).

System variables including the volume of the medium (L), ethanol concentration % (v/v), and temperature (^°C) were constantly measured using an EJA 110 differential pressure probe (Yokogawa Electric Corporation, Tokyo, Japan), an Alkosens ^® probe (Heinrich Frings GmbH & Co., KG, Bonn, Germany), and a temperature probe, respectively. The automatization of the system allows the continuous recording of data as well as testing the high reproducibility of the method. Acetic acid concentration %, (w/v) was determined by acid-base titration with 0.5 N NaOH. Viable cells concentration, the difference between total and no viable cells, were directly counted using a light microscope (Olympus BX51), a Neubauer chamber (Blaubrand™, 7178-10) with 0.02 mm depth and rhodium-coated bottom, and propidium iodide (VWR, Inc., PA, United States). Though the chamber was subdivided into 25 square groups, composed of 16 squares each, 5 square groups (0.04 mm² each) on the diagonal were used for cell counting following the method of Baena-Ruano et al. (2006) (link); samples were quantified by triplicate and standard error was calculated. These variables were exclusively measured at sampling times. The efficiency of the process was evaluated by mean acetification rate (r_A) and global production of acetic acid (p_A) which were calculated as follows: