Quantification of Cellular Sialic Acids

Total amounts of sialic acids were quantified using the periodate-resorcinol assay. Briefly, cells were washed three times with ice-cold PBS and lysed in 250 μl PBS via freeze–thaw cycles in liquid nitrogen. All samples and the standard curve were oxidised with 5 μl of 0.4 M periodic acid for >10 min on ice. Then, 500 μl of the following solution were added to each sample and mixed by vortexing: 0.6% resorcinol, 0.25 mM CuSO₄, 36% H₂0, 44% concentrated HCl. Samples were incubated at 100 °C for exactly 15 min and allowed to cool down to room temperature afterwards, before adding 500 μl of tert-butanol. To remove any particular remnants, the samples were briefly centrifuged. OD₆₃₀ was measured in triplicate in a 96-well plate and sialic acid levels were calculated from the standard curve and normalized to the total protein amount of each sample.