Victor x4 microplate reader

Acute short-term cytotoxicity (1 d) was assessed by using the standard lactate dehydrogenase (LDH) release assay as described earlier [105 (link)]. Long-term cytotoxicity was assessed by the measurement of neutral red uptake [106 (link)]. HFFs were treated with BG95 at increasing concentrations for 3 d or 7 d. Cells were incubated with 40 μg/mL neutral red solution (Merck, Darmstadt, Germany) for 3 h at 37 °C. Cells were destained with 1% acetic acid in 50% ethanol solution before neutral red signals were quantitated by photometric measurement (excitation/emission at 560/630 nm) in a Victor X4 microplate reader (PerkinElmer, Waltham, MA, USA). Assays were performed in triplicate and cytotoxicity levels are expressed as mean CC₅₀ values ± SD. The induction of apoptosis was assessed by using the commercially available BioTracker NucView^® 530 Red Caspase-3 Dye staining kit (Merck, Darmstadt, Germany) according to the instructions by the manufacturer. In brief, approximately 2 × 10⁵ cells per well were grown on coverslips in 6-well culture plates and treated with compound diluted in media beginning 1 d after seeding. At 3 d after the onset of treatment, the medium was removed and cells incubated with 5 µM NucView reagent in medium (40 min, 37 °C); subsequently, cells were fixed and stained as described in 4.6.

The antibody response to antigen in the serum of immunized mice was determined by enzyme-linked immunosorbent assay (ELISA). Briefly, twofold serial dilutions of mouse sera (from 1:50 to 1:3200) were added to antigen-precoated plates (Costar Assay Plate Half Area, Corning) and incubated for 1 h at RT prior to incubation with an HRP-conjugated secondary antibody (anti-mouse IgG, IgG₁, IgG_2a or IgG_2b; Bethyl) for 1 h at RT. To develop the reaction, plates were washed and incubated with OPD peroxidase substrate (Sigma-Aldrich), and the reaction was stopped with 3 N HCl. The optical density was read in a Victor X4 microplate reader (PerkinElmer) at 490 nm. The concentration of unknown samples was determined by interpolation in accordance with a properly generated standard curve.

As previously shown, H₂O₂ treatments can induce cell senescence and oxidative damage without significant cytotoxic effects on 3T3-L1 adipocytes^{30 (link)}.
The cytotoxicity of the different treatments was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay kit (Abcam, Cambridge, England) according to the manufacturer's instructions. Briefly, 3T3-L1 pre-adipocytes in the different experimental conditions were incubated with 50 µL of MTT Reagent and 50 µL of serum-free media for each well for 3 h at 37 °C. After incubation, the medium supplemented with the MTT reagent was removed and 150 µl of MTT solvent was added to each well. The absorbance that proportionally indicated the number of viable cells was measured at 590 nm using a PerkinElmer's Victor X4 microplate reader. The cell viability (%) was calculated as 100 × (absorbance of sample-treated cells / absorbance of the control cells)^{34 (link)}.
The MTT assay confirmed unchanged vitality in H₂O₂ treated pre-adipocytes compared to controls (see Supplementary Fig. 1).

Plasma OPN was measured by enzyme-linked immunosorbent assay (ELISA) using a kit commercially available (R&D Systems DuoSet Elisa DY6488, McKinley, MN, USA). A Victor X4 microplate reader (Perkin Elmer, Waltham, MA, USA) was used to measure the absorbance values. A calibration curve in a range of 0–1000 pg/mL range was used for sample amount determination, as suggested by the manufacturer.

Cytotoxicity of the analyzed compounds was determined by the Neutral Red dye uptake assay. A total of 1.35 × 10⁵ HFFs per well were seeded in 96-well plates 1 d prior to analysis, cultivated overnight until cells were confluent and then incubated with compounds for 7 days. The assay was performed as described previously [60 (link)] using 40 μg/mL Neutral Red (Sigma Aldrich). The quantity of dye incorporation was determined using the Victor X4 microplate reader (PerkinElmer, Waltham, MA, USA) by fluorescence measurement at 560/630 nm for excitation/emission, respectively. To assess antiviral activity of compounds, HFFs seeded in 96-well plates (1.35 × 10⁵ HFFs per well) were infected with HCMV AD169-GFP or TB40-IE2-YFP to reach 25–50% infected cells at 7 days post-infection (d p.i.). After incubation with the infectious inoculum for 1.5 h at 37 °C, compounds were added in serial dilutions. At 7 d p.i., cells were fixed for 10 min at room temperature using 10% formalin. Finally, cells were washed once with PBS followed by automated fluorescence quantitation.

Inhibition of viral replication was assayed by the use of recombinant HCMV expressing green fluorescent protein (GFP) as described previously [27 (link),103 (link),104 (link)]. In brief, HFFs were cultivated in 12-well plates (2 × 10⁵ cells per well) and infected with HCMV AD169-GFP at an MOI of 0.25 (i.e., 25% GFP-forming dose of a multi-round infection measured at 7 d p.i.). After virus adsorption of 90 min, the inoculum was replaced by medium supplemented with BG95. 7 d post-infection (p.i.) cells were lysed by the addition of 200 µL lysis buffer. Subsequently, cell suspensions were mixed and transferred to a 96-well plate. Centrifugation was performed at 3000 rpm for 15 min and clear lysates were subjected to automated GFP quantitation in a Victor X4 microplate reader (PerkinElmer, Waltham, MA, USA). All infections were performed in duplicate, GFP quantitations were performed in quadruplicate and antiviral efficacy is expressed as mean EC₅₀ values ± SD.