Versamax tunable microplate reader

Melittin (Carbosynth, Staad, Switzerland), PBS (Sigma-Aldrich, St. Louis, MO, USA), Fresh type 0 neg whole blood in citrate 96-well polypropylene plates with conical bottom, 96-well clear, polystyrene ELISA plates with flat bottom. CAPPOrigami reagent reservoirs (VWR International, Radnor, PA, USA), Microseal™ ‘F’foil (Bio-Rad, Hercules, CA, USA), Protein LoBind Eppendorf tubes, 2 mL (Eppendorf, Hamburg, Germany). Molecular Devices VersaMax^TM Tunable Microplate Reader (San Jose, CA, USA), Holm and Halby plate centrifuge B 4i (Copenhagen, Denmark).

The commercial kit Cell Titer 96^®Aqueous One Solution Cell Proliferation Assay (Promega Corporation, Madison, WI, USA) was used to assess cell viability. Briefly, BV2 microglial cells were seeded in 96-well tissue culture plates and serum-starved for 24 h. Following 30 min of pre-treatment with different doses of NaE (0–50 μg/mL) at 37 °C, cells were incubated with 0.1 μg/mL of LPS, or none, for 24 h. Afterwards, 20 µL of MTT labeling reagent was added to each well, and the plates were incubated at 37 °C for another 4 h. The formazan product formation was measured by recording the absorbance at 490 nm by using a VersaMax^TM Tunable microplate reader (Molecular Devices LLC, Sunnyvale, CA, USA). The results were expressed as optical density (OD) values, as an assessment of the number of metabolically active cells. Microglia cell viability was also assessed by trypan blue exclusion.

For protein, carbohydrate and lipid assays, absorbance was measured using a VERSAmax™ Tunable Microplate Reader (Molecular Devices, Sunnyvale, CA) set to 550, 575, 510 and 540 nm, respectively, using Softmax^® Pro v4.7 software for Windows^®. Protein was estimated using the Biuret reaction standardised against a bovine serum albumin dilution series (Sapan et al. 1999 (link)); carbohydrate using the dinitrosalicylic acid (DNS) reaction using glucose and sucrose as standards (Miller 1959 (link)); lipid using phosphoric acid–vanillin analysis colorimetry, with sunflower oil as a standard (Cheng et al. 2011 (link)). Water content of bee bread samples was determined by placing bee bread in a drying oven at 100 °C for 24 h and calculating the difference in mass between wet and dried samples. Dietary preferences and host fitness in insects often correlates with dietary protein:carbohydrate (Simpson et al. 2015 (link)) and protein:lipid ratios (Vaudo et al. 2015 (link)), we therefore, estimated these using protein/carbohydrate and protein/lipid for each sample.

To find the key domain donated to DENV adsorption, we developed a purified protein-based ELISA for testing the binding activity of each vimentin segment to DENV-2 EDIII and DENV-2. The four above-mentioned purified vimentin proteins (1 μg) were suspended in ELISA coating buffer (Beijing Dingguo Changsheng Biotechnology, China) and immobilized overnight in 96-well Immulon 2 HB plates (Thermo, USA) at 4 °C. BSA and protein-devoid systems were used as controls. The wells were washed after discarding the protein solutions. All washes were completed with 0.1 M Tris-buffered saline (TBS) at pH 7.2 with 0.05% Tween 20. Virus and antibodies were diluted by TBS; all incubations were conducted at 4 °C unless specifically stated. The wells were blocked for 1 h with 200 μL of 3% skim milk in TBS buffer and then incubated with DENV-2 (1 × 10⁴ PFU) or 1 μg of the recombinant DENV-2 EDIII protein overnight, followed by three washes and 1 h of incubation with mouse anti-DENV-2 EDIII PcAb (1:500). After washing, the wells were incubated for 1 h with peroxidase-conjugated AffiniPure goat anti-mouse IgG (1:5000) and detected by EL-ABTS Chromogenic Reagent Kit (Sangon Biotech, China). Signals were recorded at 405 nm on a VERSAmax tunable microplate reader (Molecular Devices, USA). All experiments were repeated in triplicate and reported as average and S.D.

MDA levels in tissues were evaluated using commercially available assay kits to determine the lipid peroxidation state (TBARS Assay Kit, item no. 10009055, Cayman Chemicals, Ann Arbor, MI, USA). The measuring principle is based on the reaction with thiobarbituric acid (TBA) in boiling water for 60 min in an acidic medium and the measurement of the absorbance of the reaction mixture at 532 nm (Ohkawa et al., 1979). A VersaMax Tunable Microplate Reader was used to measure the absorbance (Molecular Devices, San Jose, CA, USA). MDA concentrations in tissues were expressed as nmol MDA/mg protein.
In tissues, GPx and SOD activity were evaluated using ready-to-use assay kits. (RANSEL RS505, RANSOD SD125, Randox Laboratories Ltd., Crumlin, UK) by using an automated BS-240 VET Clinical Chemistry Analyser (Mindray, Shenzhen, China). The quantities of GPx and SOD in the tissue samples (stomach, small intestine, and large intestine) were expressed as U/mg protein.

Vero cells were cultivated in a 96-well microplate at a density of 20,000 cells/well 24 h before infection. The viral inoculum was prepared in Earle’s 199 medium with 5% bicarbonate and no addition of SFB. Cell supernatant was discarded, and 50 μL of viral suspension were added to the cell monolayer, at MOI 0.1, followed by 2 h incubation at 37°C, 5% CO₂. Then, the viral suspensions were removed, and 90 μL of supplemented Earle’s 199 medium were added to each well. After 24 h of incubation at 37°C and 5% CO₂, 10 μL of PrestoBlue Reagent (Invitrogen, Thermo Fisher Scientific, United States) were added to each well and incubated for 15 min at 37°C. Absorbance measures were acquired with SoftMax Pro 6.5 software using VersaMax Tunable Microplate Reader (Molecular Devices), at wavelength 570 nm normalized at 600 nm. Data were analyzed in GraphPad Prism software 8 with one-way ANOVA followed by Tukey’s multiple comparisons test.

To measure the concentrations of soluble Aβ oligomers, each hemibrain sample was homogenized in 8-fold volumes of homogenization medium, as described above. To quantitate total levels of Aβ42, the other hemibrain was extracted in 8-fold volumes of cold 5 M guanidine HCl plus 50 mM Tris HCl (pH 8.0) buffer and centrifuged at 20,000 g for 1 h at 4°C to remove insoluble material. Final guanidine HCl concentrations were below 0.1 M. Protein concentrations were determined by a BCA protein assay kit (Pierce). Supernatant fractions were analyzed by well-established human Aβ ELISA kits specific to oligomeric forms of Aβ (27725, IBL America, Minneapolis, MN, USA) and Aβ42 (KHB3441, Invitrogen) according to the protocols of the manufacturers. Optical densities at 450 nm of each well were read on a VersaMax tunable microplate reader (Molecular Devices, Sunnyvale, CA, USA), and sample Aβ oligomer and Aβ42 concentrations were determined by comparison with the respective standard curves. Aβ oligomer and Aβ42 concentration values were normalized to total brain protein concentrations and expressed in picograms and nanograms per milligram of total protein, respectively.