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11 protocols using amplex red

1

Redox Cycling Compound Assay

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The assay was adapted from a previously described protocol to assess redox cycling of compounds in the presence of reducing agents (51 (link)). 13 nl of compounds (or DMSO control) were pin-transferred into 2.5 μl HBSS (Thermo Fisher; containing 1.26 mm CaCl2, 0.49 mm MgCl2, 1 g/l d-glucose) in black 1,536 well plates. Compound fluorescence was measured immediately (READ 0) using a ViewLux uHTS microplate imager (PerkinElmer) equipped with Ex: 525/20 and Em: 598/25 filters. 2.5 μl of a 2X Amplex Red solution [100 μm Amplex Red (Cayman Chemical, Ann Arbor), 200 μm DTT (Thermo Fisher) and 2 U/ml horse radish peroxidase (Sigma-Aldrich); diluted in HBSS and protected from light] were added to each well. Fluorescence was measured after a 15 min incubation at room temperature (READ 1), using ViewLux settings identical to READ 0. Activity was calculated using corrected fluorescence values (READ 1 minus READ 0), which were compared with control samples (negative = vehicle; positive = 46 μm walrycin B).
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2

Membrane Lipid Dynamics Analysis

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10-Acetyl-3,7-dihydroxyphenoxazine (Amplex red)(Cayman Chemicals); Choline oxidase (MP Biomedicals); horseradish peroxidase; mβCD (Acros organics); 1,2-dioctanoyl-sn-glycero-3-phosphocoline (C8-PC) (Avanti Polar Lipids Inc.). Amplex red was first dissolved in dimethyl sulfoxide to prepare 10 mM stock solutions and stored frozen at −20 °C, protected from light for up to 6 months. C2C12 cells (ATCC CRL-1772). Corning Cellgro DMEM (Fisher Scientific, MT10013CV). Cellgrow FBS (VWR, SH3039603). Goat α-rabbit IgG (VWR PI-1000) 1:2,000; α-PLD2 (E1Y9L, Cell Signaling #13891), 1:150; α-PtdIns(4,5)P2 (Echelon, Z-P045), 1:200. Donkey α-mouse Cy3B antibodies were a gift from Manasa Gudheti. PLD1 (VU0359595) and PLD2 (VU0364739) inhibitors (500 nM) were a gift from Alex Brown.
Cluster analysis was performed using VutaraSRX cluster analysis tool over multiple images. GraphPad Prism was used to determine significance for raft size; CTxB (n=1,907), CTxB+mβCD (n=344), PIP2 control (n=3,076), PIP2+mβCD (n=778). Cell differentiation assay was performed single blind on images taken from multiple dishes (n=4 for each condition). All numbers are reported as mean±s.e.m. unless otherwise noted. As all samples were found to have a normal distribution, Student's t-test was used to determine significance with resultant P-values as reported.
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3

Hemin-Assisted G-Quadruplex RNAzyme Assay

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The rPS2.M G quadruplex was heat-cycled to with NaClO4 to attain proper configuration: heat to 80 °C and cool to 10 °C at 1-min intervals every 10 °C. The sample was then incubated with hemin at room temperature for 5 min. The G quadruplex/hemin RNAzyme was allowed to react with 10-Acetyl-3,7-dihydroxyphenoxazine (Amplex Red, Cayman Chemical Company, Cat#10010469) and Hydrogen peroxide for 5 min. Fluorescence readings were taken on a SpectraMax Gemini EM microplate reader (Molecular Devices; SoftMax Pro software V 5.4) with an excitation wavelength of 565 nm and an emission wavelength of 585 nm. Data was normalized to a negative control containing Amplex Red but no rPS2.M/hemin. Working solutions contained 5 µM rPS2.M, 1 µM hemin, 5 mM phosphate buffer pH 7.4, 5 µM Amplex Red, 10 µM H2O2, 0.5% DMSO, and variable NaClO4.
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4

Quantifying Lipid Metabolism Markers

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Free fatty acids and glycerol released in the media were measured on a BioTek Synergy H1 microplate reader as previously detailed49 (link). Free fatty acid release was determined using a NEFA-HR kit (Fujifilm) adapted to fluorescence detection with Amplex Red (10-Acetyl-3,7-dihydroxyphenoxazine, Cayman Chemical) and the fluorescence was read for Resorufin with excitation at 554 nm and emission at 593 nm. Glycerol release was determined using Free Glycerol Reagent (Sigma) and the absorbance was measured at 540 nm.
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5

Adipocyte Lipolysis Assay Protocol

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Doxycycline inducible WT PNPLA3-cherry and PNPLA3 I148M-cherry mouse brown adipocytes (BAs) were seeded on either 12 well or 24 well plates as noted. After 48 hours of induction medium, the cells were grown in differentiation media for 24 hours, followed by media containing no doxycycline or amount of doxycycline specified for a total of 4 days. After one week of differentiation, the cells were stimulated with specified treatment of either vehicle, SR-3420, or isoproterenol at the indicated concentration in H-KRBB buffer for 1 hour. Free fatty acid release was determined using WAKO kit (Wako Pure Chemicals Industries) adapted to fluorescence detection with Amplex Red (Cayman Chemical). Glycerol release was determined using glycerol reagent from triglyceride determination kit (Sigma).
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6

Adipocyte Lipolysis Assay Protocol

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Doxycycline inducible WT PNPLA3-cherry and PNPLA3 I148M-cherry mouse brown adipocytes (BAs) were seeded on either 12 well or 24 well plates as noted. After 48 hours of induction medium, the cells were grown in differentiation media for 24 hours, followed by media containing no doxycycline or amount of doxycycline specified for a total of 4 days. After one week of differentiation, the cells were stimulated with specified treatment of either vehicle, SR-3420, or isoproterenol at the indicated concentration in H-KRBB buffer for 1 hour. Free fatty acid release was determined using WAKO kit (Wako Pure Chemicals Industries) adapted to fluorescence detection with Amplex Red (Cayman Chemical). Glycerol release was determined using glycerol reagent from triglyceride determination kit (Sigma).
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7

Measuring Cellular Oxidative Stress

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HEPA1–6 (ATCC CRL-1830), were cultured in DMEM with 10% fetal bovine serum (FBS; Gibco) at 37 °C and 5% CO2. Cells were seeded at approximately 300k cells/12 well. Steady-state levels of cellular H2O2 production were measured using the horseradish peroxidase-linked Amplex Red fluorometric assay (Cayman) per manufacturer’s instructions. Steady-state levels of intracellular levels of O2•− were measured using dihydroethidium and MitoSOX (Thermo Fisher) and fluorescence was recorded using a microplate reader (Tecan).
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8

Zinc Acetate-Mediated Synthesis

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Chemicals and materials for this work were as follows: zinc acetate
dihydrate (J.T. Baker); pure ethanol (Koptec); ethanolamine (Alfa
Aesar); Al (RD Mathis); aurochloric acid trihydrate (Beantown Chemical);
Amplex Red (Cayman Chemical); horseradish peroxidase (Amresco/VWR);
sodium phosphate monobasic and dibasic (Mallinckrodt Chemical); Decon
90 (Electron Microscopy Sciences); isopropanol (Fisher Chemicals);
hydrogen peroxide, 35% w/w (BDH/VWR); and ammonium hydroxide 28–30%
w/w (BDH/VWR).
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9

H2O2 Quantification in Fibroblasts

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H2O2 release was measured through the conversion of Amplex red reagent by peroxidase to produce the red-fluorescent oxidation product, resorufin [22] (link). Following treatment, IPF fibroblasts were washed twice, and incubated with a reaction mixture (100 µM Amplex red [Cayman Chemical, Ann Arbor, MI], 5 U/ml horseradish peroxidase, 1 mM HEPES in Hank’s Balanced Salt Solution without phenol red). After a 90 minute incubation, signals were measured with excitation and emission wavelengths at 544 and 590 nm, respectively. H2O2 concentrations were calculated by plotting against a standard curve.
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10

Quantifying Hydrogen Peroxide Levels in Neuronal Cells

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Cells were seeded on black 96-well plates (2 × 104 Neuro2a cells/ well, 3 × 104 SH-SY5Y cells/ well) and treated as described above.
For determination of H2O2 accumulating in the medium during the treatment period, 50 µl conditioned cell culture supernatant of each well were transferred to a new 96-well plate and supplemented with 5 µM Amplex Red (Cayman Chemical, Ann Arbor, USA) and 0.01 U/ml HRP (Thermo Fischer Scientific, Schwerte, Germany). A potential interference between the added agents and the HRP-catalyzed reaction between Amplex Red and H2O2 was analyzed by repeating this experiment in a cell-free system utilizing freshly prepared DMEM/0.1% FCS + phospholipids (10 µM) or solvent in the presence of supplemented H2O2 (0.000007%).
For measuring the H2O2 freshly released by pretreated cells, the supernatant was removed and 50 µl of Amplex Red reaction mixture (5 μM Amplex Red and 0.01 U/ml HRP in phenol red-free DMEM/0.1% FCS) was added to each well.
The fluorescence signal of resorufin was determined at an excitation wavelength of 530 ± 17 nm and an emission wavelength of 590 ± 17 nm for 16 min (120 s (seconds) intervals) at 37 °C under light exclusion in a Safire2 Fluorometer (Tecan, Crailsheim, Germany). The increase of fluorescence over time was calculated for each well and used for further data analysis.
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