Proteogreen gglu

GGT activity was measured using the MaxDiscovery GGT Enzymatic Assay Kit (Bioo Scientific) according to the manufacturer’s protocol. BMDMs were pre-incubated for 1 h with 10 μM acivicin before plating cells on 25 μg/ml fibrin-coated 6-well culture plates. For in vivo GGT activity assay, spinal cord tissues from peak disease of MOG_35-55 EAE or LPS-injected substantia nigra area at 12 h were prepared. Tissues were homogenized in 0.1 M Tris-HCl and centrifuged at 13,000g for 30 min at 4 °C. The supernatant was collected and assessed for GGT activity by measuring the absorbance at 405 nm with a microplate reader as described above. The GGT activity in IU/l was calculated following the manufacturer’s protocol by multiplying the average increase in absorbance at 405 nm over 10 min. GGT activity was also measured with fluorescent probe, ProteoGREEN-gGlu (Goryo Chemical), according to the manufacturer’s protocol. BMDMs were plated on 96-well, black μ-clear-bottom microtiter plates (Greiner Bio-One) pre-coated with 25 μg/ml fibrin. BMDMs were incubated with ProteoGREEN-gGlu with acivicin or GGsTop (diluted at threefold concentrations from 0.01 μM to 8.3 μM). The fluorescence intensity (excitation/emission filter pairs of 488 nm/520 nm) was measured using a microplate reader as described above.