Microplate reader

Measurements were performed with the lipophilic potentiometric dye 3,3′-dipropylthiadicarbocyanine iodide (diSC3(5)). E. coli bacteria (ATCC 25922) were grown overnight in M9 minimal media and diluted 1000-fold in M9 and grown for 5 h at 37 °C until an OD₆₀₀ of 0.5. Bacteria were then washed with 5 mM sodium HEPES, 2 mM EDTA buffer (pH 8) and resuspended in the same buffer at an OD₆₀₀ of 0.05. The bacterial samples were then incubated with 0.4 µM diSC3(5) in black Greiner CELLSTAR^® (655090) 96-well plates for 60–90 min, until a stable reduction of fluorescence was achieved. Following nanostructure formation treatment test compounds were then added to the stabilized bacterial solutions to a final concentration of 250 µg/ml and the changes in the fluorescence signal was monitored continuously using a CLARIOstar® BMG LABTECH microplate reader (excitation, 622 nm; emission, 670 nm). 1% TritonX-100 was used as a positive control. Results displayed are representative of three independent experiments conducted.

ROS production was measured using L-012-enhanced chemiluminescence, as previously described (To et al., 2017 (link)). Inflammatory cells isolated from the BAL were seeded into a 96-well OptiView plate (5 × 10⁴ cells/well) with Dulbecco’s Modified Eagle’s Medium (DMEM; Thermofisher, United States) containing 4.5 g/L of glucose, 110 mg of sodium pyruvate and 10% Fetal Bovine Serum (FBS; Sigma-Aldrich, United States), and allowed to adhere for 3 h prior to starting the assay. Cells were then washed with warm 37°C Krebs-HEPES buffer and exposed to a Krebs-HEPES buffer containing L-012 (10⁻⁴ mol/L) (WAKO Chemicals) in the absence (i.e., basal ROS production) or presence (stimulated ROS production) of the protein kinase C (PKC) and NADPH oxidase activator, phorbol 12,13-dibutyrate (PDB; 10⁻⁶ mol/L) (Sigma-Aldrich, United States). The same treatments were performed in blank wells (i.e. with no cells), which served as controls for background luminescence. All treatment groups were performed in triplicate. Photon emission [relative light units (RLU)/s] was detected using the BMGlabtech microplate reader (CLARIOstar, Germany) and recorded from each well for 1 s over 60 cycles. Individual data points for each group were derived from the average values of the three replicates minus the respective blank controls.

ROS production was quantified using L-O12-enhanced chemiluminescence. Cells isolated from the BAL were seeded into a 96-well OptiView plate (5 × 10⁴ cells/well) with Dulbecco's modified Eagle's medium (Thermo Fisher) containing 4.5 g/L of glucose, 110 mg of sodium pyruvate, and 10% fetal bovine serum (FBS; Sigma-Aldrich), and allowed to adhere for 3 h before starting the assay. Cells were washed in Krebs-HEPES buffer at 37°C and then exposed to a Krebs-HEPES buffer containing L-O12 (10⁻⁴M) in the absence (i.e., basal ROS production) or presence (stimulated ROS production) of the protein kinase C and NADPH oxidase activator phorbol dibutyrate (10⁻6 M). The same treatments were performed in blank wells (i.e., with no cells), which served as controls for background luminescence. All treatment groups were performed in triplicate. Photon emission (relative light units/s) was detected using the BMG Labtech microplate reader (CLARIOstar, Germany) and recorded from each well for 1 s over 60 cycles. Individual data points for each group were derived from the average values of the three replicates minus the respective blank controls.

Blood samples were collected by venepuncture of the median cubital vein and EDTA vacutainers (BD Vacutainer®). Samples were rested for 30 min and then centrifuged at 4000×g for 10 min at 4 °C. Plasma was subsequently pipetted into aliquots and immediately stored at -80 °C until time of analysis. Plasma concentrations of NfL and CAF were determined by ELISA (#OKCD01380, Aviva Systems Biology, San Diego, CA, USA and #ab216945, Abcam, Cambridge, UK, respectively) according to previously described protocols [18 (link), 19 (link)]. Plasma NCAM levels were also measured using a commercially available ELISA (#ab119587, Abcam, Cambridge, UK) according to the manufacturer’s instructions. Briefly, 100 μL of standards and diluted sample (100-fold dilution) were added to the pre-coated microplate and incubated at 37 °C for 90 min. Then, 100 μL of NCAM detector antibody was added to each well and incubated at 37 °C for 60 min. Next, the microplate was washed three times and then 100 μL of Avidin–Biotin-Peroxidase Complex working solution was added into each well and incubated at 37 °C for a further 30 min. The microplate was washed five times and then Add 90 μL of TMB was added into each well and incubated at 37 °C for 20 min in the dark. Lastly, 100 μL of TMB stop solution was added into each well and the measurements were read at 450 nm (CLARIOStar BMG Labtech Microplate reader).

ARPE-19 cells were seeded in 96-well plates and treated with various concentrations of SMARTPOOL ZKSCAN3 siRNA (25, 50, 100, μM) (Dharmacon, Lafayette, CO, USA) for 24, 48, 72, and 96 h. We used a range of concentrations and time points to evaluate dose and time-dependent effects of the siRNA on the viability of the RPE. The medium with different concentrations was then removed and cells were incubated with 20 µL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (5 mg/mL) for 4 h at 37 °C. The optical density was measured at 490 nm using a Microplate Reader (BMG Labtechnologies, Cary, NC, USA) after dissolving the formazan with 0.5% DMSO.

Normal rat kidney fibroblast cells (NRK-49F) were obtained from the American Type Culture Collection (Rockville, MD, USA) and cultured in Dulbecco’s Modified Eagle Medium (Gibco BRL, Gaithersburg, MD, USA) containing 10% fetal bovine serum (Gibco BRL), 100 μg/mL penicillin, and 100 μg/mL of streptomycin. The cells were incubated in a 95% air- and 5% CO₂-humidified atmosphere at 37 °C. The NRK-49F cells were seeded in a 96-well plate at 5 × 10³ cells/well and allowed to attach for 24 h. Then, the medium was replaced with serum-free media. After 24 h of serum starvation, the cells were treated with serum-free media containing Mel (0.5, 1, and 2 μg/mL) for 30 min, followed by a treatment with 5 ng/mL of TGF-β1 for 8 h. The cells were then washed with PBS, and the MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide/PBS) was added to each well. The plates were incubated for 4 h at 37 °C. Finally, the MTT-containing medium was removed by aspiration, and 100 μL of dimethylsulfoxide solution was added to each well. The absorbance value was measured at 540 nm using a microplate reader (BMG labtechnologies, Mornington, Germany).

PPAR-γ activity was measured using a PPAR-γ transcription factor assay kit (ab133101, Abcam, Cambridge, MA, USA) according to the manufacturer’s protocol [36 (link)]. Briefly, nuclear extracts from the cells were prepared using a nuclear extraction kit (Abcam, Cambridge, MA, USA). Then, the nuclear proteins were added to 96-well plates precoated with a specific double-stranded DNA sequence containing the peroxisome proliferator response element. The plates were incubated at 4 °C overnight and then washed five times with wash buffer. The plates were incubated with the specific primary anti-PPAR-γ antibody at room temperature for 1 h, followed by incubation with the horseradish peroxidase-conjugated secondary antibody. After washing the plates, we added a developing solution and incubated them for 20 min, after which we added the halting solutions. Absorbance was measured at 450 nm using a microplate reader (BMG Lab Technologies, Offenburg, Germany).