Ensight multimode plate reader

The RBL-2H3 cells and astrocytes were plated at the density of 4 × 10³ and 5 × 10³ cells/well respectively in a 96-well plate. After the appropriate treatments at 24 and 48 h, the medium was removed and the cells incubated with a 1 mg/mL solution of Thiazolyl Blue Tetrazolium Bromide (MTT; Merck, Milan, Italy), at 37 °C, for 1 h and a half. The dissolution of the crystals was carried out with 200 µL of DMSO per well, and the absorbance was read at 590 nm using the EnSight Multimode Plate Reader (Perkin Elmer) [38 (link)].

At 12 dpi, mice were i.v. injected with 100 μL of 3% Evans blue in PBS. Three hours later, mice were anesthetized and perfused with ice-cold PBS; SCs were then dissected. The tissue was then cut into small pieces with scalpels, and Evans blue was extracted by incubating the tissue in deionized formamide at 56°C overnight. After centrifugation at 16,100 g at room temperature (RT) for 15 minutes, supernatant from the homogenate was carefully taken, filtered through a 70-μm cell strainer (Fisher Scientific), and transferred to a fresh 1.5-mL Eppendorf tube. The optical densities of formamide extracts from SC tissues at 620 nm and 740 nm were measured on an EnSight Multimode Plate Reader (PerkinElmer). Evans blue absorbance was corrected for turbidity by subtracting the optical density at 740 nm from the total optical density at 620 nm (60 (link)).

Cell viability was assessed using the Cell Counting Kit-8 assay (CCK-8; Dojindo, Kumamoto, Japan). GBM cells (4 × 10³ cells/well) were seeded into 96-well plates and cultured at 37°C. After 12 h the medium was replaced with 100 uL of culture medium containing different concentrations of GG (Sigma-Aldrich; MO, USA) or vehicle control (dimethyl sulfoxide, DMSO; Sigma-Aldrich, MO, USA). At 24 and 48 h after dosing, GBM cells were incubated with 10 μL of CCK-8 reagent in 100 μL of serum-free DMEM at 37°C for an hour. The absorbance at 450 nm was measured using EnSight Multimode Plate Reader (PerkinElmer; Singapore).

Compound plates were prepared by dispensing 2 µL of test compound dissolved in DMSO into wells of a 96-well black tissue culture treated plate (Corning). Each compound was tested in a 10-point serial 3-fold dilution. Wells with DMSO (final concentration of 0.8%) or a compound at a concentration at which viral replication was completely inhibited were used as assay Min_E and Max_E control, respectively. HEp-2 cells (ATCC) were infected in cell suspension at an MOI of 0.001, distributed into the wells, and incubated at 37 ˚C, 5% CO₂ for 1 h. Plates were then centrifuged at 200 × g for 10 min and cells were overlaid with a 125 µL/well of 1% methylcellulose solution in MEM containing 2% FBS and 100 U/mL penicillin-streptomycin. Plates were incubated at 37 ˚C, 5% CO₂ for 3 days. Following incubation, the overlay was removed and cells were fixed with acetone. Plaques were detected by incubating cells with monoclonal antibodies 143-F3-1B8 RSVαF (Sino Biological) and 34C9 RSVαN (Sino Biological), followed by incubation with goat anti-mouse Alexa 488 antibody (Invitrogen). The plates were read on a Perkin Elmer Ensight multimode plate reader using brightfield and Alexa 488 settings with data analysis using a custom plaque algorithm. Antiviral EC₅₀ values were determined using a 4-parameter logistic fit based on the Levenberg-Marquardt algorithm. Model 205, 4-Parameter Logistic.

The plasmin activity was assayed in cell supernatants collected from HUVEC and HNEpC (seeded in a 24-well plastic plate at 1.5 × 10⁵ cells per well) as reported in [16 (link)]. Beside the solutions of interest, the mesoglycan was also used at 300 µg/mL for 24 h as positive control and the apotrinin (Sigma-Aldrich; St. Louis, MO, USA), as a serine protease inhibitor, administered at 30 µM, 10 min before reading. The samples were analyzed through the Plasmin Activity Assay kit (Abcam, Cambridge, UK) following the manufacturer’s instructions and adapted to cell media. Particularly, the standard curve was created, starting from a plasmin concentration of 0 ng/well (in 50 µL of standard volume/well) to 75 ng/well. A total of 50 µL of each experimental point was used for reading in triplicate. A total of 50 µL of reaction mix (plasmin assay buffer + plasmin substrate) was added to standard and sample wells. The output fluorescence was evaluated as Ex/Em = 360/450 nm at the EnSight Multimode Plate Reader (PerkinElmer; Waltham, MA, USA) in a kinetic mode (a reading each 2 min from 10 to 20 min of plate incubation at 37 °C). The amount of plasmin was calculated, as previously reported.