Gemini xps

Detection of NETs was assessed with cfDNA and CitH3, considered one of the most specific markers of NET formation [58 (link)]. As found in previous studies, NETs can predict more severe episodes of COVID-19 pneumonia and estimate different clinical trajectories [59 (link)].
Plasma samples were mixed with a monoclonal mouse anti-histone biotinylated antibody (Component 1, Cell Death Detection ELISAPLUS) in a streptavidin-coated plate (Component 9). A rabbit polyclonal anti-histone-H3 (citrullinated R17 + R2 + R8) (ab81797; Abcam Inc., Waltham, MA, USA) antibody was used in a second step. Detection was carried out with a peroxidase-linked antibody (GE Biosciences, Barcelona, Spain). A pool of samples from normal subjects was used to normalize values. It was included in all microplates, being expressed as individual absorption values.
To determine cfDNA, plasma was diluted 1:10 with phosphate-buffered saline (PBS (in mmol/L: NaCl 137, KCl 2.7, Na₂HPO₄ 10, KH₂PO₄, pH 7.4)) and mixed with an equal volume of 1 mm SytoxGreen (Invitrogen, Carlsbad, CA, USA). Fluorescence was determined in a fluorescence microplate reader (Gemini XPS; Molecular Devices, Sunnyvale, CA, USA). A calibration curve was generated with calf thymus DNA (Invitrogen) in PBS. More detailed information is provided elsewhere [60 (link)].

The quantification of the level of total PA in the vitreous fluid was done according to the manufacturer’s instruction (Cat No: DVC00, Cayman Chemical Company, Ann Arbor, MI). The PA assay kit uses the fluorescence based methods wherein the presence of peroxidase, H₂O₂ reacts with ADHP (10-acetyl-3,7-dihydroxyphenoxazine) to give highly fluorescent compound known as resorufin. The total lipids were first extracted from the each vitreous samples and were dried under a gentle stream of nitrogen. The sample was resuspended in 1% triton X-100 and was ready to use for the analysis or storage. After adding 10 μL of each samples in 96 well plate, the reaction was initiated by adding 40 μL of lipase and the plate was incubated at 37°C for 1 hour. The detector mixture was prepared as detailed and 50 μL of which was added to all well and was kept for another 30 minutes at room temperature. The fluorescence was read using an excitation wavelength of 530 nm and emission wavelength of 585 using Gemini XPS fluorescence microplate reader (Molecular Devices, LLC, Sunnyvale, CA). Each assay was performed in duplicate. Using the linear regression curve equation for standard, the concentration for each sample was calculated.

The Aβ_25–35 self-aggregation assay was performed following the method described by Naldi et al. [13 (link)]. First, peptide was pretreated with HFIP for 1 day at 22 °C to obtain non-amyloidogenic conformation. Various doses of phytoestrogens in 34.5 mM phosphate buffer (pH 7.4) with 17.5% MeCN were mixed with a 0.1 mM monomeric Aβ_25–35 solution in a 1:29 ratio (v/v) and incubated overnight at 4 °C. After incubation, the reaction mixture was supplemented with 0.025 mM thioflavin-T in 50 mM glycine–NaOH buffer (pH 8.5). The fluorescence emission was monitored at 490 nm with excitation at 446 nm using the Gemini XPS (Molecular Devices, Sunnyvale, CA, USA). Curcumin was used as the standard [13 (link)].

The LAL enzymatic activity was determined using 4-methylumbelliferyl oleate (4-MUO; MilliporeSigma) as substrate as described previously (51 (link)). Proteins were extracted from HD1A myeloid cells, which had been treated with Lalistat2 or DMSO for 72 hours. Protein concentrations were determined by Pierce BCA protein assay kit (Thermo Fisher Scientific). Eighteen micrograms of protein was added to 0.567 mM substrate solution (0.567 mM 4-MUO, 0.15 M sodium acetate/0.01% Tween-80, pH 5.5, 1% [vol/vol] Triton X-100). Reaction was incubated at 37°C for 30 minutes, and then terminated by addition of 100 μL of 0.75 M Tris, pH 8.0. A standard curve was prepared ranging from 0 to 100 nM 4-methylumbelliferone (MilliporeSigma). Fluorescence was measured on a Gemini XPS plate reader (Molecular Devices) at 355 nm excitation and 460 nm emission. Data were analyzed using the SoftMax program. Assays were linear within the time frame of these assays, and less than 10% of substrate was cleaved. One unit is 1 mmol of 4-MUO cleaved per minute under standard assay conditions.

The GSH assay kit (S0073, Beyotime) was adopted to examine the SCs of the GSH level according to the manufacturer’s protocol. By reference to the manufacturer’s instructions, DCFH-DA (S0033, Beyotime) was used for total ROS level measurement. SCs (5000 cells/well at concentration) were plated in a 96-well microplate and processed as indicated. Next, the DCFH-DA (10 μmol/L)-loaded cells were placed for 30 min away from light (37 °C) and gently cleaned 3 times in PBS. The fluorescence microscope (Nikon, Tokyo, Japan) together with a microplate reader (Gemini XPS, Molecular Devices, Gothenburg, Sweden) were employed to detect total ROS for the fluorescence intensities.

SCE cells were prepared by the same method used for TEER measurements as described above. SCE cell monolayers were then stimulated with 1, 5, or 25 μmol/L of K-115, Y-27632, or fasudil. A tracer, fluorescein isothiocyanate (FITC)-dextran (average molecular weight, 4000; Sigma-Aldrich), was simultaneously applied at 50 μmol/L to culture well basal compartments. Culture medium was collected from the apical side for fluorescence measurements at 60 min after the addition of tracer with an equal volume of the culturing medium added to replace the removed medium. FITC-dextran concentrations in collected medium were measured using a multimode plate reader (Gemini XPS; Molecular Devices, LLC, Sunnyvale, CA) with an excitation wavelength of 490 nm and an emission wavelength of 530 nm. The fluorescence intensity of normal medium was measured and used as the background concentration in each experiment.