Multiscan fc microplate photometer

Superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) were analysed as antioxidants^{(18 (link),19 )}. In the peritoneal fluid samples taken during the surgical intervention, nitric oxide (NO)^{(20 (link))} from reactive oxygen radicals; lipid peroxidation product malondialdehyde (MDA); and myeloperoxidase (MPO), which is mainly present in acute inflammation, were examined using a Shimadzu UV Spectrophotometer UV-1800 (Japan) before and after the treatment^{(20 (link),21 (link),22 )}. In addition, the measurement of VEGF-A, which is one of the important regulator peptides defined in angiogenesis, was examined using a Multiscan FC Microplate Photometer (Thermo Scientific, Finland) device using Rat ELISA (Wuhan EIAAB Science Co., LTD. Wuhan, China) kit^{(23 )}.

The protein contents of the isolated EVs were measured using a Pierce Micro-bicinchoninic acid (BCA) Protein Assay (Thermo Scientific, Berlin, Germany), according to the manufacturer’s instructions. Absorbance was measured at 562 nm using a Multiscan FC Microplate Photometer (Thermo Scientific, Berlin, Germany).

Cell proliferation was evaluated using the WST-8 assay kit (Nacalai Tesque, Kyoto, Japan). Two thousands of MCF-7 or TamR cells per well were cultured in 96-well plates and 10 μL of WST-8 solution was added to each well at the indicated time points after transfection. Cells were further incubated for 2 h at 37 °C in a 5% CO₂ incubator. The absorbance was measured at 450 nm with Multiscan FC Microplate Photometer (Thermo Fisher Scientific, Rochester, NY, USA).

Levels of IgG antibodies against SARS-CoV-2 antigens (N-protein, RBD and S1) were estimated by xMAP SARS-CoV-2 Multi-Antigen IgG Assay and xMAP SARS-CoV-2 IgG Control Kit (Luminex Corp.) using the serum samples of 223 COVID-19 patients (mild (n=20), mild-moderate (n=41), moderate (n=71), severe (n=60), ICU (n=31) according to the manufacturer’s instructions. Acquisitions were performed using a MAGPIX instrument operated with xPONENT software version 4.2 (Luminex Corp.). Assay’s sensitivity and specificity characteristics: for ≤7; 8-14; >14 days from symptom onset positive percent agreement was 71.1% (55–83% 95% Cl); 80.0% (58–92% 95% Cl); 98.1% (90–100% 95% Cl) respectively, and negative percent agreement was 100% (99%-100% 95% Cl). Serum IgM and IgA in samples from all 444 COVID-19 patients were measured using a COVID-19 ELISA IgM+IgA kit (Vircell) following the manufacturer’s instructions. Optical density measurements were performed using a Multiscan FC microplate photometer operated with Skanit Software version 4.1 (Thermo Scientific). Assay’s sensitivity in patients 5 days after a positive PCR result was 88%, and specificity in samples from healthy pre-pandemic donors was 99%.

Cytotoxicity of bLF and the LF peptides toward HeLa cells was analyzed by monitoring mitochondrial activity. For this, approximately 5 × 10⁴ HeLa cells were seeded in a 96-well plate and cultured serum-free for 16 h. Cells were washed twice with PBS. A serial dilution of bLF and each peptide was made in PBS (0–25 µM). Cells we were incubated at 37 °C under 5% CO₂ for 1 h. Maximal cytotoxicity was induced by adding 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA). Cells were washed twice with PBS and incubated in PBS containing 0.5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, ThermoFisher Scientific, Bleiswijk, the Netherlands) for 2 h and washed again. The MTT-crystals that were precipitated in the cells were resuspended in 100% dimethylsulfoxide (DMSO, Sigma-Aldrich). Absorption was measured at 570 nm with 630 nm for background correction using a Multiscan FC microplate photometer (ThermoFisher Scientific,). In parallel, cytotoxic effects of bLF and the peptides on Hela cells were analyzed by measuring the lactate dehydrogenase (LDH) release using an LDH-cytotoxicity kit (Abcam, Cambridge, UK) according to the manufacturer’s instructions (Sijbrandij et al. 2017 (link)). The experiments were performed in duplicate and repeated three times.

Immune response to food ingredients was assessed by a solid-phase, non-competitive, indirect enzyme-linked immunosorbent assay (ELISA) using a commercial reagent kit for a semiquantitative enzyme immunoassay of allergen-specific IgG antibodies (LLC NPO Immunoteks, Stavropol, Russia). This kit uses monoclonal anti-IgG antibodies included in the peroxidase conjugate that are capable of detecting antibodies of the immunoglobulin G class in human serum/plasma, which have an affinity for allergens sorbed on the surface of a polystyrene tablet. The analysis was performed according to the recommendations of manufacturer. The kit contained 2 polystyrene 96-well plates with food antigens immobilized on the surface. One kit was designed to determine the content of specific IgG in eight test samples for 22 allergens (Table 4).
ELISA was performed on a Multiscan FC microplate photometer (Thermo Scientific, Waltham, MA, USA), and the results interpreted according to the manufacturer’s instructions (Table 5).