Wallac 1420 arvo

HPF is a novel reagent that directly detects certain highly reactive oxygen species (hROS)34 (link). Although HPF itself has little fluorescence, it selectively and dose-dependently reacts with hROS, such as ·OH and peroxynitrite, and shows strong fluorescence. HPF (Goryo Chemical, Hokkaido, Japan) was added to H₂-dissolved solution (H₂ group) or normal solution (control) at a final concentration of 5 μM. As in the ESR experiment, in 10 ml of the solution in the 50-ml plastic test tubes, US oscillation without irrigation and aspiration was performed for 10 seconds at 30% power. Immediately after US, fluorescence of the samples was measured by a plate reader (Wallac 1420 ARVO; PerkinElmer, Waltham, MA) with excitation at 485 nm and emission at 535 nm. All measurements were performed 4 times.

In a 96-well
plate, Jurkat cells (2.0 × 10⁴ cells/well) were incubated
in the presence of 2-APB (50 μM), ER-000444793 (10 μM),
RuRed (10 μM), Dynasore (10–30 μM), Mdivi-1 (10–30
μM), CID1067700 (1–10 μM), Z-VAD-fmk (30 μM),
Nec-1 (30 μM), and 3-MA (5 mM) in 10% FBS RPMI medium (50 μL)
for 1 h at 37 °C under 5% CO₂, and then solutions
of 4 (10 μM) and celastrol (2 μM) in 10%
FBS/RPMI 1640 medium (50 μL) were added. The final concentrations
of 4 and celastrol were 5 and 1 μM, respectively.
The resulting solutions were incubated at 37 °C under 5% CO₂ for 1 and 3 h (4) or 24 h (celastrol), after
which a 0.5% MTT solution in PBS (10 μL) was added to each well.
After incubation for 4 h at 37 °C under 5% CO₂, a
10% SDS in 0.01 N HCl aqueous solution (100 μL) was used as
a formazan lysis solution and the resulting solutions were incubated
overnight under the same conditions, followed by the measurement of
the absorbance at 570 nm using a multilabel counter, Wallac 1420 ARVO
(PerkinElmer).

Lipase activities in the culture supernatants of the WT, Δ145138, and 145138OE lines were measured using 4MU-palmitate (Sigma-Aldrich) as a substrate^{20 (link)}. Briefly, 10 μl of the culture supernatant was mixed with 180 μl of 50 mM Tris-HCl, pH 8, containing 2 nmol of 4MU-palmitate. To assess the effect of inhibitors, BEL or MAFP, 145138 was pre-incubated with several concentrations of BEL for 10 min, or MAFP for 5 min, then mixed with 4MU-palmitate. The reaction mixture was kept at 37 °C for the appropriate time, and 4MU released was measured using a Wallac 1420 ARVO fluorescence microplate reader set at 355 nm Ex/460 nm Em (PerkinElmer).