Ez read 400 elisa microplate reader

vWF-antigen levels and vWF-activity were determined turbidimetric assays on a Siemens BCS XP system applying appropriate reagents (vWF Ag and INNOVANCE vWF Ac kits, respectively, all by Siemens Healthcare). Normal ranges of vWF-antigen and vWF-activity were 70–120% and 61–179%, respectively, in patients with all blood types; normal vWF-activity was 46–146% in patients with blood type O.
For measurement of ADAMTS13 activity, citrate plasma samples of 50 patients were kept in aliquots at −20°C before analysis in the central laboratory. ADAMTS13 activity was determined applying the Technozym ADAMTS-13 Activity ELISA kit (Technoclone, Heidelberg, Germany) on a Biochrom EZ Read 400 ELISA Microplate Reader at 450 nm according to the manufacturer's instructions.

MTT cytotoxicity assay was performed as described previously [38 (link)]. Briefly, B16F10 cells were cultured at 96-wellmicroplates (5000 cells/well). After 24 h the cells were treated with different concentrations of the extracts (in control cells an appropriate amount of DMSO was added). The MTT solution (MTT, Sigma-Aldrich) (1 mg/mL in serum free, phenol red free medium) was added 48 h after the addition of test extracts. After 4 h, the MTT solution was discarded and isopropanol was added to dissolve the formazan crystals. Absorbance was recorded at 570 nm using a microplate reader (EZ Read 400 ELISA microplate reader, Biochrom, Cambridge, UK). Survival of non-treated cells was set to 100%.

The NO inhibitory activity was performed using the same procedure as previously reported [19 (link)]. RAW 264.7 (American Type Culture Collection, Manassas, VA, USA) were seeded at 4 × 10⁴ cells/well in 96 well plates suspended in 100 μL DMEM supplemented with 10% FBS and incubated at 37 °C 5% CO₂ overnight. Cells were incubated with 1 µg/mL LPS for 1 h and treated with various concentrations of sample compounds and vehicle control (DMSO) for 24 h. After 24 h, the NO production inhibitory was determined in the culture supernatant using the Griess reaction by adding 100 µL of Griess reagent in a 96 well plate for 10 min. The determination of nitric oxide was measured at 570 nm with Biochrom EZ Read 400 ELISA microplate reader (Biochrom Ltd., Cambridge, United Kingdom). Additionally, the data were presented as IC₅₀ which was calculated with GraphPad Prism 6.0 software. Indomethacin was used as a positive control of NO production inhibitor with an IC₅₀ value of 19.61 µM.