Elx800 bio tek

The total flavonoid content was determined using the AlCl₃ method (Aluminum trichloride) as described by [29 (link),30 (link)]. FCe 4 mg in 1 mL DMSO, 10 gm aluminum chloride in 100 mL distilled water (dH₂O), 1M potassium acetate in distilled water (98.15 g/100 mL), and 1 mg quercetin in 1 mL methanol were used to make the stock solution. A 96 well plate was filled with 20 µL of FCe. With the help of a micro-pipette, 10 µL of 10% aluminum chloride was added to FCe in a 96 well plate and mixed. 10 µL potassium acetate (1 M) was added. Finally, 160 µL dH₂O was added to each well, resulting in a final concentration of 20 µL in each well, which was thoroughly mixed. Negative control of 20 μL methanol was used, while a positive control of 50, 25, 20, 10, 5, and 2.5 µg/mL Quercetin was used. The plate was incubated at 37 °C for 39 min after mixing. A microplate reader (ELx800 Bio-Tek, BioTek^® Instruments, Inc., Winooski, VT, USA) was used to read the samples at 415 nm. Quercetin equivalent µL/mg was used to calculate the results. Three times the experiment was repeated.

Free radical scavenging activity (FRSA) was measured using 2,2-diphenyl-1-picrylhydrazyl (DPPH) for the determination of antioxidant potential, as described by [67 (link)]. The stock reagent solution was prepared by dissolving 3.2 mg of DPPH in 100 mL of methanol and stored in a refrigerator until use. Briefly, 180 μL of 2,2-diphenyl-1-picrylhydrazyl (DPPH) reagent was added to the entire row of wells containing 20 μL of the samples to obtain the final concentrations of 200 μL. The OD was recorded at 517 nm using a microplate reader (ELx800BioTek, BioTek Instruments, Colmar, France) after 1 h of incubation in the dark at room temperature. The antioxidant potential of each biological sample was calculated as % DPPH discoloration, calculated by the following formula:
where Abc = absorbance of the control and Abs = absorbance of the sample

aPS/PT IgG and IgM were measured in 96 well microplates by ELISA (Aeskulisa Serine-Prothrombin-GM, AESKU.DIAGNOSTICS, Wendelsheim, Germany) following the procedures indicated by the manufacturer. Optical density was obtained by reading the microplates at 450 nm and 620 nm, for background subtraction, in a microplate reader (Bio Tek ELx800, Agilent, Santa Clara, CA, USA). Sera from 79 healthy donors were used to setup the cut-off at 99th percentile for aPS/PT IgG and IgM, which resulted in a value of 17 U/mL, nearly equal to the 18 U/mL established by the manufacturers. Patients above 18 U/mL were deemed as positive for aPS/PT. Those patients between 14 U/mL and 18 U/mL were considered as low–middle positive.

The complete OptiMem or CHECM, or complete OptiMem collected from cultures of control or transfected SC-derived myoblasts (48 h after transfection) or CHECM conditioned with control or transfected SC-derived myoblasts presence for 24 h, was analyzed using Human VEGF SimpleStep ELISA kit (ab222510, Abcam), accordingly to manufacture’s instruction. 562 nm absorbance was measured using a microplate reader BioTek ELx800 (Agilent) with Gen5 software (Agilent). Three independent experiments were performed in duplicates. The average results for each experiment were shown on graphs. The graphs were performed using GraphPad 9 software (Prism, www.graphpad.com ).

Melting points were determined in open capillaries on a FALC Instrument SRL360D. TLC (Silica Gel 60 F254) from Merck (Beijing, China) was used to check the compounds’ purities. Elemental analyses were carried out by using Euro EA 3000 (EUROVECTOR, Pavia, Italy) instrument. The infrared spectra of the Schiff bases were recorded on FT-IR (Bruker Optics GmbH & Co. Ettlingen, Germany) with an ATR model (ALPHA 200488) in the frequency range of 4000–550 cm^–1. NMR spectra were recorded in DMSO-d₆ and CDCl₃ solvent on a BRUKER AV-500 MHz NMR (Bruker Beijing Scientific Technology, Beijing, China). A high-resolution multinuclear FT-NMR spectrometer was used in the presence of SiMe₄ (internal standard) at δ = 0 ppm at 25 °C.
The sbsorbance of α-amylase and α-glucosidase activities (at 540 and 405 nm, respectively) were measured on a microplate reader (BioTek Elx-800, Agilent Technologies, Santa Clara, CA, USA). An InnuPREP Blood DNA Mini Kit (Analytik Jena) (AJ Innuscreen GmbH, Berlin, Germany) was used for the extraction of genomic DNA from human blood. A PDA spectrophotometer (Agilent 8453) Agilent Technologies, Berkshire, UK, along with a quartz optical cell (with an effective absorption path of 1 cm), was used for DNA interaction studies of the synthesized Schiff bases.