Cytation 3 cell imaging multi mode plate reader

Nile Red Staining was performed as previously described (Benova et al., 2022 (link)). Cells were cultured in polystyrene flat-bottom 96-well tissue culture-treated black microplates (BRANDplates^®, cellGrade™, Brand, DE). Nile Red dye (Merck) working solution was prepared from a stock solution of 1 mg/ml. Cells were washed with PBS (Gibco). Dye was added directly to the cells (5 μg/ml in PBS), incubated for 10 min at room temperature in the dark, and then, washed twice with PBS. The fluorescent signal was measured using a Cytation 3 cell imaging multimode plate reader (BioTek) using an excitation of 485 nm and an emission of 572 nm. The fluorescent signal of Nile Red stain was normalized to cell viability signal measured by Cell Titer-Blue Assay Reagent (Promega) mentioned previously.

Nile Red Staining was performed as we previously described [20 (link)]. It is a direct stain for the detection of intracellular lipid droplets by fluorescence microscopy. Cells were cultured in polystyrene flat-bottom 96-well tissue culture-treated black microplates (BRANDplates®, cellGrade™, Brand, Wertheim, Germany). Nile Red dye (Merck, Darmstadt, Germany) working solution was prepared from a stock solution of 1 mg/ml. Cells were washed with PBS (Thermo Fisher Scientific, Waltham, MA, USA). Dye was added directly to the cells (5 μg/ml in PBS), and incubated for 10 min at room temperature in the dark, then washed twice with PBS. Fluorescent signal was measured using a Cytation 3 cell imaging multimode plate reader (BioTek, Winooski, VT, USA) using excitation of 485 nm and emission of 572 nm. The fluorescent signal of Nile Red stain was normalized to cell viability signal measured by Cell Titer-Blue Assay Reagent (Promega, Madison, WI, USA) mentioned above.

The superoxide anion scavenging activity of blank HSA-PEI NP’s, HSA-PEI-pSOD1 Np’s, and HSA-PEI-SF-pSOD1 Np’s at different time intervals (10, 24, and 48 h) were evaluated with a commercially available SOD assay kit (Sigma Aldrich). Briefly, a sample solution (20 μL, i.e., 2 µg SF and 300 ng pSOD1) was added with 200 μL of WST-1 (2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)- 2H-tetrazolium, monosodium salt) working solution in a 96 well plate. With the addition of 20 μL enzyme (xanthine oxidase) solution, the reaction was initiated and preceded at 37°C for 20 min. Finally, the inhibition activity was measured by taking the absorbance at 450 nm as compared to the control using a Cytation 3 cell imaging multi-mode plate reader (Biotek).

The levels of total cytosolic ROS, total mitochondrial ROS, and the specific level of mitochondrial superoxide anion radicals were assessed using the fluorescent dyes H₂DCF-DA, DHR, and the Red Mitochondrial Superoxide Anion Indicator (MitoSOX), respectively. The iso-WT and triple tau-mutant iPSCs were plated each with 8–12 replicates into Cellartis DEF-CS COAT-1-coated black 96-well cell culture plates at a density of 20,000 cells per well. The next day, the iPSCs were loaded in the dark with a final concentration of 10 µM of DCF for 20 min, with 10 µM of DHR for 15 min, or with 5 µM of MitoSOX for 2 h under agitation at room temperature. Afterward, the plates were washed twice with HBSS before measuring. During the incubation, H2DCF-DA and DHR are oxidized to dichlorofluorescein (DCF) and cationic rhodamine 123, respectively, generating fluorescent products. The fluorescence signals were detected at 485 nm (excitation)/535 nm (emission) using the Cytation 3 Cell Imaging Multi-mode Plate Reader (BioTek). MitoSOX is oxidized specifically by mitochondrial superoxide, forming a highly fluorescent product, which was detected at 531 nm (excitation)/595 nm (emission). The fluorescence intensities were proportional to cytosolic ROS levels, mitochondrial ROS levels, and superoxide anion radicals in mitochondria. The data were normalized on the protein content.

The E. coli reporter strain was grown overnight in 5 mL LB medium containing 30 μg/mL kanamycin and 100 μg/mL ampicillin at 37 °C. Overnight culture was diluted 1:100 in 20 mL of fresh LB medium supplemented with proper antibiotics and grown at 37 °C until OD₆₀₀ reached 0.5. L-arabinose (Sigma-Aldrich, St. Louis, MO, USA) and 3O-C12HSL (Sigma-Aldrich) were added into bacterial culture to a final concentration of 0.1% (w/v) and 10 μM, respectively. Subsequently, 150 μL of the reporter strain culture was added to each well of a 96-well white plate (Corning). Next, 2.5 mM stock compound dissolved in DMSO was added to the well with a final concentration of 100 μM. The compounds used for screening were obtained using an in-house compound library [43 (link),44 (link),45 (link),46 (link),47 (link)]. Plates were incubated at 30 °C for another 4 h with shaking. The bioluminescence and the absorption at 600 nm (OD₆₀₀) were recorded on a Cytation 3 cell imaging multi-mode plate reader (BioTek).

BBB transit assays were performed as described above, the inserts were removed from 12-well plates and immersed twice in HBSS+ (Hanks balanced salt solution containing 1% glutamine, 1% PenStrep, 140 mg/liter CaCl₂, 100 mg/liter MgCl₂, 100 mg/liter MgSO₄, and without phenol red), after which both sides were fixed with 4% formaldehyde–PBS (10 min, room temperature [RT]). The fixed inserts were washed in HBBS+ twice and both sides stained with CellMask Deep Red Plasma membrane stain (Thermo Fisher Scientific) (1:10,000 in PBS; 10 min, RT) and washed twice in HBBS+. The inserts were then excised from the plastic housing using 10-mm-diameter skin biopsy specimen punches and placed monolayer side up on a bubble of mounting media on a microscope slide. Another few microliters of mounting media and a coverslip were then applied. After the mounting media had cured, the membrane was viewed on a Zeiss LSM 510 confocal laser scanning system. To image cells directly, we used Fluoroblok plates and media without phenol red. Wells were washed once with HBSS+ and fixed as described above, and the lower part of the insert was imaged without removal from the plate using a Cytation3 cell imaging multimode plate reader (Bio-Tek Instruments, Inc.).

Quantification of triglycerides in liver and serum used the Triglyceride Colorimetric Assay Kit (Cayman Chemical, Ann Arbor, MI, USA). Collected tissue samples were stored frozen at −80 °C. For the colorimetric analysis, the samples were processed according to the manufacturer’s recommendations. Here, 2-mL liver tissue homogenates were produced from pre-weighed fragments of frozen samples (350–400 mg). Triglyceride concentration in the serum was measured without diluting the samples, and the liver homogenates were diluted five-fold. Absorbance was measured by using a Cytation 3 Cell Imaging Multi-Mode Plate Reader (Biotek, Winooski, VT, USA).