Black μ clear bottom microtiter plates

BMDMs or PBMCs were incubated in RPMI containing 5 μM DHE (Invitrogen) for 30 min. 100,000 cells/well were plated on 96-well black μ-clear-bottom microtiter plates (Greiner Bio-One) precoated with 25 μg/ml fibrin. 20 μg/ml 5B8 or IgG2b (UCSF Monoclonal Antibody Core clone LTF-2) were added in fibrin-coated wells 2 h before plating cells. BMDMs were incubated with 300 μM apocynin (Calbiochem) for 1 h or 5 μg of anti-CD11b (M1/70, eBioscience) for 30 min before plating. Cells were incubated on fibrin for 24–48 h, fixed with 4% PFA for 10 min, and DHE fluorescence was detected at 518_nm/605_nm using SpectraMax M5 microplate reader. Cells were quantified by DAPI counterstaining. In mice, in vivo DHE administration and detection was performed as described^{15 (link)}.

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.