Minimax 300 imaging cytometer

For the adhesion assay, opto-TLR4 PANC-1 cells were transferred to non-adhering plates (30,000 cells/ well; S-Bio, Hudson, NH, USA; MS-9096UZ) and immediately treated with LPS, or light and different concentrations TAK-242 or PAR.
For continuous measurement, the “electrical cell-substrate impedance sensing” (ECIS) model 9600Z was used. Therefore, 9W20idf PET plates (ibidi, Gräfelfing, Germany; 72098), precoated with 40 µg/mL neutralised rat tail collagen type I (Thermo Fisher Scientific, Vienna, Austria; A1048301), were overlaid with the pre-treated cells, and cell attachment and spreading were assessed by continuous resistance measurements for 20 h. For endpoint measurement, cells were plated and allowed to attach on 96-well cell culture plates (pre-coated with collagen I) for 30 min (attachment assay), rinsed twice with 1xPBS and adhering cells were automatically counted using the Mini Max 300 Imaging Cytometer (Molecular Devices, LLC, San Jose, CA, USA; 5022671).
For the proliferation assay, cells (3000 cells/well) were treated with light or left untreated for 0–72 h and cells were automatically counted using the Mini Max 300 Imaging Cytometer (Molecular Devices, LLC, San Jose, CA, USA; 5022671) every 24 h.

ROS production was detected by using the oxidation-sensitive fluorescent probe, CM-H₂DFDA (Molecular Probe, Invitrogen). The cells were first pre-incubated with CM-H₂DFDA (2 µM) in PBS for 30 min, followed by exposure to rotenone in complete cell culture medium for 2 h. The medium was then replaced with HBSS and fluorescence (Ex 485 nm/Em 520 nm) analysed by Spectramax i3x plate reader. To ensure a representative readout of the fluorescent adherent cells, 37 different points were read in each well using the well scan function of the plate reader. Equal cell number in each well were verified by staining the nuclei with DRAQ5 (nuclear staining, 1:500; Thermo Fisher) for 30 min at room temperature, and cell number were counted by the spectramax i3x plate reader equipped with a microscopic module (MiniMax300Imaging Cytometer, Molecular Devices). For flow cytometric analyses, the cells were treated (CM-H₂DFDA pre-incubation and rotenone exposure) in eppendorph tubes for reasons as described in 2.5. When using the efflux-pump inhibitor probenicid (1 mM), it was used during the CM-H₂DFDA staining and rotenone exposure.

Cytotoxic effects of DON (0.8, 1.6, 3.1, 6.3, 12.5, and 25 µM) in astrocytes and microglia were analysed after 24 h exposure with CellTox^TM Green Dye (1:2000). The dye was added to the cells as described by the manufacturer, and fluorescence signals (Em: 485 nm/Em: 520 nm) were quantified with a Spectramax i3x plate reader using the well scan function by reading 32 different points/well in all wells. The cytotoxicity data were normalised by the total cell numbers, which were determined by staining with the nuclear stain DRAQ5 (1:500, 30 min) and counting with the Spectramax i3x plate reader equipped with a microscopic module (MiniMax300imaging cytometer, Molecular Devices), Ex: 625 nm, Em: 713 nm.