Microplate reader

BEPS-IA treated cells were lysed in solubilization buffer (50 mM Tris, pH 7.4; 1% Igepal; 150 mM sodium chloride; 1 mM EDTA) with protease inhibitors (1 μg/mL aprotinin; 1 μg/mL leupeptin; 1 μg/mL pepstain; 1 mM phenylmethylsulfonyl fluoride (PMSF); 1 mM sodium orthovanadate; 1 mM sodium fluoride) for 30 min at 4 °C, and insoluble materials was removed by centrifugation at 12,000× g for 10 min at 4 °C. The supernatant was collected and the protein concentration was determined by Sigma Diagnostics Micro Protein Determination Kit and Dynex Microplate Reader (Dynex Technologies, Chantilly, VA, USA). Equal amounts of protein (60 mg) were loaded in each lane and separated by 12% SDS-PAGE. The proteins were transferred to Immobilon-p transfer membranes. The membranes were blocked with 3% non-fat dry milk in TBS containing 0.1% Tween 20, and then incubated at room temperature for 1 h in the presence of each antibody (Anti-Bax antibody, Anti-P53 antibody and anti-Bcl-2 antibody). The membrane was washed three times with 0.1% Tween 20 in PBS and stained with HRP-linked anti-mouse IgG secondary antibody. β-Actin was used as internal control. The ECL system (Amersham Biosciences, Buckinghamshire, UK) was used for detection [40 (link)].

SAT2 antigens were incubated at different temperatures (4 °C, 40 °C or 47 °C) for 10 minutes and their stability was analyzed by double antibody sandwich ELISA using llama single-domain antibody fragments (VHH domains), termed M377F VHH, against intact FMD viral particles (146 S infectious virions). M377F VHH was provided by the Central Veterinary Institute of Wageningen UR, AB Lelystad, The Netherlands^{18 (link)}. The wells of a 96-well microtitre plate were incubated overnight with VHH domains in carbonate/bicarbonate buffer (50 mM, pH 9.6; Sigma Aldrich, UK) at a concentration of 0.5 mg/l. Test samples serially diluted in VHH buffer (1% skimmed milk; 0.05% Tween; 0.5 M NaCl; 2.7 mM KCl; 2.8 mM KH₂PO_4; 8.1 mM Na₂HPO₄, pH 7.4.) were added to the wells and incubated for 1 hour at 37 °C. Next, the biotinylated VHH-M377F, diluted in VHH buffer at a concentration of 0.1 mg/l, was added and incubated for 1 hour at 37 °C. Plates were washed with PBS-Tween (0.05% Tween 20) and bound biotinylated VHH-M170F was visualised by incubation with streptavidin-HRP (Dako, UK) for 1 hr at 37 °C followed by addition of o-phenylenediamine dihydrochloride (Sigma, UK) for 15 minutes at room temperature. The reaction was stopped by addition of 1.25 M sulphuric acid and quantified at 492 nm using a Dynex microplate reader (Dynex Technologies, UK). ELISAs were performed twice in triplicate.

For MTT test,[15 ] MDCK II and MDCK II-Best1 cells were plated at initial concentration of 5 × 10⁴ cells per well, A549 and PRE-1 were plated at initial concentration of 6 × 10⁴ cells per well.
After 24 h of incubation at 37 °C (310.15 K), the cells (at a density of 5 × 10⁴ cells per well) were incubated for two hours with different concentrations of sPLA₂ (from 0.5 to 2.5 × 10⁻⁶ mol/L⁻¹). The cells not treated with sPLA₂ served as a control and were used to normalize the viability data. After certain time intervals, MTT solution was added to each well at a final concentration of 0.5 × 10⁻³ kg L⁻¹ and the plates were incubated at 37 °C (310.15 K) for four hours. The MTT formazan product was dissolved by addition of 1.1 × 10⁻⁴ L acidified 2-propanol (in 4 × 10⁻² mol L⁻¹ (0.04 N) HCl) to each well. The absorbance was detected at 5.4 × 10⁻⁷ m (540 nm) using Dynex microplate reader (Dynex Technologies, USA). Cell survival rate was calculated as ratio of the (absorbance of the treated wells)/(absorbance of the control wells) × 100%.
To establish the kinetics of interaction sPLA₂‑cells and appearing of eventual morphological changes, cells were incubated with sPLA₂ (1.5 × 10⁻⁶ mol L⁻¹) for different times, visualized with an inverted microscope (XDS-2) and stained for death cells with 0.5% Trypan blue.

For the cell cytotoxicity test, hDPSCs purchased from Lonza (PT-5025; Basel, Switzerland) at passage 8 were used. The cytotoxicity of the synthesized powder was evaluated using the MTT assay (Ahn et al., 2020 (link)). To determine the proper concentration of powder, a pre-test was conducted with four different amounts of powder (125, 250, 500, and 1,000 μg/well). hDPSCs were seeded at a density of 1 × 10⁴ cells/well in a 24-well culture plate. The cells were cultured in the material extraction full media-containing α-MEM (Gibco), 10% FBS (Merck), 1% penicillin-streptomycin (Gibco), and 5 μg/ml plasmocin (InvivoGen, San Diego, CA) in a 5% CO₂ incubator at 37°C for 24 h. The culture medium in each well was replaced with 100 μL fresh culture medium containing 10 μL MTT solution (5 mg/ml MTT in sterile phosphate-buffered saline; Sigma-Aldrich, St. Louis, MO, United States) and incubated for 4 h at 37°C and 5% CO₂. The medium was replaced with 100 μL of dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO, United States) for 5 min at 37°C. The absorbance was subsequently measured at 570 nm using a microplate reader (Dynex, Lincoln, United Kingdom). After determining the proper powder concentration, the MTT assay was conducted using the same process for 1, 2, 3, and 7 days. All results were obtained from three independent experiments and control wells were also prepared with full media.

Release of DPP4 or leptin from human primary adipocytes were detected in the cell culture supernatants by ELISA (R&D Systems, Wiesbaden, Germany). Cells were cultured in either growth or differentiation media. After renewal of media, cells were further incubated for 24 h and finally, cell supernatants were collected for analysis. The assays were performed according to the manufacturer’s protocol and measured in a microplate reader (Dynex, Chantilly, VA, USA) against a standard curve.

Cells were grown in 96-well plates at a density of 5 × 10³ cells/well in a 200μl volume. For the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, 20μl of MTT solution (5 mg/ml in PBS) was added into each well and incubated at 37°C for 4 h. Then MTT solution was removed, 150μl of dimethyl sulfoxide (DMSO) was added into each well. After 10 min of shaking at room temperature to completely dissolve formazan crystals, the absorbance was detected at 570 nm using a Microplate Reader (Dynex Technologies, U.S.A.).