Imark reader

The 5-HT2AR binding assay was carried out as previously reported [18 ] using (as capture antigen) an 18-meric linear synthetic peptide, Q….N, (LifeTein Inc., Hillsborough, NJ) having amino acid sequence identical to that of the second extracellular loop region of the human 5-HT2AR. A 1/25th dilution of the protein-A eluate fraction of plasmas was added to each well in duplicate. Autoantibody-antigen binding was detected with the use of peroxidase conjugated, goat-anti-human IgG (Sigma Chem Co, St Louis, MO) and appropriate substrate solution [18 ]. Color development was monitored at 490 nm using an IMark reader (Biorad).

Pellets cultured for 1, 7 or 14 days were digested overnight at 60 °C in 100 μL papain digestion buffer (pH = 6.4) containing 0.1% papain, 10 mM EDTA-disodium salt (both from Merck), 100 mM sodium acetate, and 5 mM L-Cysteine·HCL (both from Sigma-Aldrich). Following digestion, samples were diluted 1:8 in water to measure the GAG content or samples were diluted 1:6 in Tris-EDTA (TE) buffer to measure DNA content. The GAG content was measured by adding 200 μL dimethylmethylene blue (DMB) solution (0.05 mM DMB, 41 mM NaCl, 45 mM glycin and pH = 3.0) to 40 μL papain-digested sample (pre-diluted 1:8 in water) and absorbance was measured at 590 nm using an iMark Reader (Bio-Rad). To determine the DNA content, PicoGreen^® stock solution (ThermoFisher Scientific) was diluted 1:200 in TE buffer (10 mM Tris-HCl, 1 mM EDTA and pH = 7.5) and 50 μL of this solution was mixed with 50 μL papain-digested sample (pre-diluted 1:6 in TE buffer). After 5 minutes dark incubation at room temperature, fluorescence was measured at 485/520 nm (excitation/emission) with a CLARIOstar (BMG Labtech, Offenburg, Germany) using DNA obtained from human HEK293T cells to set the standard curve.

The recombinant extracellular domains of the receptors DR5 (100 ng/well), VEGFR1 or VEGFR2 (50 ng/well) (R&D Systems, Minneapolis, MN, USA) were immobilized on ELISA plates overnight at 4 °C in 0.1 M carbonate–bicarbonate buffer (pH 9.4). The plates were washed three times with PBST (phosphate-buffered saline + 0.05% Tween), and wells were blocked by 2% BSA in PBST for 1 h at 37 °C. After blocking, dilutions of DR5-B, HRH–DR5-B or SRH–DR5-B (in 3 replicates) at concentrations from 0.032 to 2500 nM were added, and the plates were incubated for 1 h at 37 °C. Captured ligands were detected by subsequent incubation with monoclonal antibodies to TRAIL (MAB375, R&D Systems, Minneapolis, MN, USA) and anti-mouse polyclonal goat IgG conjugated with horseradish peroxidase (HAF007, R&D Systems). Unbound antibodies were washed 3 times with a PBST buffer, and color was developed by OPD (o-phenylenediamine dihydrochloride) colorimetric substrate. After 15 min of incubation with the substrate at 37 °C, the reaction was stopped by a 1 N H₂SO₄ solution. The optical density was determined at 450 nm by iMark reader (Bio-Rad, Hercules, CA, USA). Dissociation constant (K_D) values were calculated by GraphPad Prism 8 software (GraphPad Software Inc., San Diego, CA, USA), using the nonlinear regression option in the XY analysis section.

Cells were seeded in a 96-well plate one day prior to treatment with IAld or I3AA. RAW cells and HUVEC were treated for 24 h. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was then added in accordance with the protocol for MTT Assay for Cell Viability and Proliferation (Millipore Sigma). Absorbance was read at 595 nm after 4 h using a Bio-Rad iMark reader. The absorbance relative to the vehicle control was reported (Mean ± SEM) and correlates with relative cell viability [58 (link)].

Cells were seeded at 3 × 10⁵ cells/well in 6-well cell culture plates, and culture for 24 h. Culture were then stimulated exposed to vehicle control (DMSO) or various concentrations of PAC (0, 1, 2.5, 5 and 10 μM) for 24 h. At the end of the stimulation period, the culture medium of each condition was supplemented with MTT solution at 0.5 mg/mL then incubated for 3 h at 37 °C in the dark. The cells were washed, then 1 ml of 0.04 N HCl in isopropanol was added to the culture wells, followed by an extended 15-min incubation. Finally, 200 µl of the reaction mixture (in triplicate) was transferred to the wells of a 96-well flat-bottom plate and the absorbance was measured at 550 nm using iMark reader (Bio-Rad). The cell viability (percentage) was determined by using the following formula: % of cell viability = [(OD_550 nm (treated cell)/(OD (control cell)] × 100. This experiment was repeated 7 times. To support the cell viability assay we performed lactate dehydrogenase (LDH) activity measurement using culture supernatants. Supernatants were collected from each condition after exposure or not of the cells to PAC for 24 h. The LDH activity was measured by means of an LDH kit from (Sigma-Aldrich), as outlined in our previous work^{28 (link)}. The experiment was repeated 4 independent times.