Spectramax m 3 multi mode microplate readers

RAW264.7 cells were cultured in 24-well plates. To measure β-hexosaminidase activity, 100 μL culture medium was incubated with 100 μL of 1 mM 4-Methylumbelliferyl N-acetyl-β-d-glucosaminide (Sigma, USA) in 0.2 M citrate buffer (0.1 M citric acid, 0.1 M sodium citrate, pH 4.5) for 1 h at 37 °C. Reactions were quenched by addition 200 μL of 0.2 M Tris base. Cells pellets were lysed with 1% Triton X-100, and then treated as above. β-hexosaminidase activity was indicated by the fluorescence of the product that was measured by SpectraMax M-3 Multi-Mode Microplate Readers (Molecular Devices, USA) with an excitation wavelength of 365 nm and an emission wavelength of 460 nm. The release percentage of β-hexosaminidase was calculated as follows: [β-hexosaminidase in culture medium / (β-hexosaminidase in culture medium + total β-hexosaminidase in pellet)] ×100%.