Microfluor 1 plate

The following components were added to a black 96-well round-bottom Microfluor 1 plate (Thermo Scientific): reaction buffer (10 mM HEPES, pH 7.9, 50 mM NaCl, 0.01 v/v % Tween-20), enzymes, cofactors, additives (vide infra), and 3-fold compound dilutions from DMSO stocks (1 v/v % final DMSO concentration) to a final volume of 90 μL. Each well was then mixed thoroughly by manual pipetting, followed by the immediate addition of 10 μL of ARK(Me3)STGGK peptide substrate. Typical final concentrations are 50 μM iron(II) ammonium sulfate hexahydrate, 500 μM ascorbate, 2 mM NAD⁺, 0.0252 U formaldehyde dehydrogenase (FDH) per reaction, 1 μM KDM4C (residues 1–352), 50 μM peptide substrate, and variable concentration of α-KG (2.5–50 μM). The reaction was monitored by measuring the change in fluorescence intensity over time on a SpectraMax M5e plate reader with an excitation wavelength of 350 nm and emission wavelength of 460 nm.

Each rat CSF sample (100 μL; rat CSF 25 μL was diluted with 75μL PBS) or human CSF sample (100 μL) was incubated with JC1 (2 μL of 50 μM in 50% DMSO) for 20 min at 37 °C in 96-well MIcrofluor 1 plate (Thermo Fisher Scientific). JC1 dye exhibits potential-dependent accumulation in mitochondria, indicated by fluorescence emission shift from green (Ex 485 nm/Em 516 nm) to red (Ex 570 nm/Em 590 nm). Back ground fluorescent signal from each sample (100 μL containing 2 μL of 50% DMSO) was subtracted from JC1 fluorescent value and mitochondria membrane potential was determined by the red/green fluorescent ratio after top-read measurement in SpectraMax M5 microplate reader.