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Whole cell lysate (20 μl) was combined with 80 μl of 0.05 N H2SO4 (hydrolysis reagent) and incubated at 80°C for 60 min. If you have any sort of inquiries pertaining to where and ways to use Supplier of sialic acid powder as Raw Material for beverages, you could contact us at our own web page. Samples were briefly centrifuged at 14,000 revolution/min (16,000 g), after which 20 μl of 1 M NaOH (neutralization reagent) was added and the mixture centrifuged again at 14,000 revolution/min. Afterwards, wells were washed 3 times, 100 µl of substrate solution was added for 30 min at RT and the optical density (OD) was measured at 405 nm by an ELISA reader (TECAN Infinite 200 PRO). The wells were washed 3 times and then 100 µl of a specific alkaline phosphatase labelled antibody for terminal complement complex (TCC, C5b-9; provided by the manufacturer) was added and incubated for 30 min at RT.