Plate spectrophotometer

We used the thiobarbituric acid reactive substances (TBARS) method to measure lipid peroxidation in the hippocampus of the rats, following the manufacturer’s recommendations (Invitrogen, Thermo Fisher Scientific, MA, USA). In brief, 300 μl of hippocampus homogenates were treated with 0.6 N trichloroacetic acid and clarified by precipitating the interfering proteins. Thus, the absorbance of all samples was measured at 450 nm and 560 nm using a plate spectrophotometer (Bio-Tek, Winooski, VT, USA), as indicated by the manufacturer. This assay recognizes oxidized lipids by a reaction between malondialdehyde (MDA) and thiobarbituric acid in the presence of heat. The reaction produces TBARS, which are detected and quantified by a spectrophotometer and compared with a standard curve.

Levels of RANTES, I-CAM-1 and TGF-β1 were determined as per the manufacturer’s instructions described briefly below.
The standards and appropriately prepared plasma samples (100 µL) were added to the designated wells. The plate was sealed and incubated for 1.5 hours at 37°C. Following the incubation period, the plate contents were discarded followed by the addition of biotinylated detection antibody (100 µL) to each well and the plate incubated for 1 hour at 37°C. The plate was then washed three times using an automated plate washer (BioTek Instruments, Inc., Winooski, VT, USA) followed by the addition of horseradish peroxidase (HRP) conjugate (100 µL) to each well. The plate was incubated for an additional 30 minutes at 37°C. The plate was washed a further five times as described above followed by the addition of 3,3’,5,5’-tetramethylbenzidine (TMB) substrate reagent (100 µL) and a further 15-minute incubation period at 37°C, protected from light. The reaction was stopped by the addition of 50 μL stop solution and the optical density (OD) read at a wavelength of 450 nm using a plate spectrophotometer (BioTek Instruments Inc.). The concentration of the analyte present in each sample was determined using the appropriate generated standard curve and the results are presented as nanograms (ng)/mL.

The DBCA protocol employed in this study was performed as described previously [37 (link),38 (link)]. Briefly, HEK293 cells (ATCC, CRL-1573) expressing ΔCR PrP were seeded on 24-well plates at approximately ~60% confluence (day 1). On day 2, cells were treated with 500 μg/mL of Zeocin, and with different concentrations of CPZ, Fe(III)-TMPyP, dynamin inhibitors or vehicle controls (volume equivalent). Medium containing fresh antibiotic/compounds was replaced daily until day 3, after which the medium was removed, and cells were incubated with 1 mg/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma Aldrich, St. Louis, MO) in PBS for 30 min at 37°C. After carefully removing MTT, cells were resuspended in 500 μL of DMSO, and cell viability values obtained by a plate spectrophotometer (BioTek Instruments, VT, USA), measuring absorbance at 570 nm.

Cells were seeded on 24-well plates at ∼60% confluence. Compounds at different concentrations or vehicle control (0.1% DMSO, volume equivalent) were added after 48 h, medium was replaced the second day, and then removed after a total of 48 h of treatment. Cells were incubated with 5 mg/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma Aldrich) in PBS for 15 min at 37 °C. After carefully removing MTT, cells were resuspended in 500 μL DMSO, and cell viability values obtained by a plate spectrophotometer (BioTek Instruments, VT, USA), measuring absorbance at 570 nm.

The embryonic alpha-1-acid glycoprotein (0.1 µg/mL) was dissolved in 0.1 M carbonate buffer, pH 9.5, containing 0.15 M NaCl, and 150 µl was added to the first well and was two-fold serially diluted in the same buffer in each well of a polystyrene 96-well ELISA microtiter plate (Maxisorp, Nunc), and then incubated at 4°C overnight. After incubation, the plate was washed three times with the buffer containing 0.01 M Tris-HCl, pH 7.5, 0.15 M NaCl, 0.05% Triton X-100 (TBS-T) and then three times with water. After that, 160 µL of bovine serum albumin (1 µg/mL) in TBS-T was added to each well to block non-specific binding sites. The plate was then incubated for 1 hour at room temperature and washed as described above. 150-µL aliquots of the samples containing chimeric lectin CmAP/MBL-AJ with concentration 100 µg/mL were added to each well. TBS-T was used as negative control, and interaction of MBL-AJ with adsorbed antibodies under the same conditions served as a positive control. After that, the plate was kept for 1 h at room temperature and washed again. Then 150 µL of pNPP in the buffer for AP assay was added to each well. After 2–5 min of incubation, 100 µL of 2 M sodium hydroxide was added to stop the reaction. Absorbance of the resultant solution was measured at 400 nm with a plate spectrophotometer (Bio-Tek Instruments, USA).

Tumor cells were seeded into 96-well plates with 1000 cells in each well, and cultured in a 37 °C incubator overnight. In the experimental wells, 1 μg/ml doxycycline was added to induce KANK1 expression. On the second day, cells were washed twice with PBS, and incubated with MTT (0.5 mg/ml) for 4 hours. Then, crystalized purple-colored formazan in cells was dissolved by adding 100 μl DMSO per well. Optical density at 595 nm was recorded using a plate spectrophotometer (Bio-TEK). For each cell line or experimental condition, at least 3 replicates were performed at each time point.

Standard ELISA plates (Nunc Maxisorp^TM, ThermoFischer Scientific, Massachusetts, USA) were coated with 1 μg/mL cytokine (IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F) in PBS pH 7.4 overnight. Plates were washed three times in wash buffer (PBS supplemented with 0.05% Tween20, SigmaAldrich, Missouri, USA) and tapped dry. Diluted antibody was added to the relevant wells and incubated for 1 h at room temperature. Plates were washed three times and a goat IgG-horseradish peroxidase conjugated antibody with specificity for human Fc (Jackson ImmunoResearch, Pennsylvania, USA) was added to each well. Plates were incubated for 1 h at room temperature. Plates were washed for a final time before the addition of TMB (3,3′,5,5′-tetramethylbenzidine) Stabilized Substrate (Promega, Southampton, UK) for 8 min, after which an equal volume of stop solution (1 M H₃PO₄) was added to each well. Absorbance was then measured at 450 nm using a plate spectrophotometer (Biotek Instruments).