Measurement of labile heme concentration in PL fluid samples was performed in 96-well plates as previously described (22 (link)). Briefly, 5 µL of an appropriately diluted PL fluid sample was added to 95 µL of HBSS buffer containing 0.75 µM Apo-Horseradish Peroxidase (Apo-HRP) (BBI Solutions, Gwent, UK) and incubated for 10 min at 4°C. Simultaneously, hemin standards ranging from 0.25 to 2.5 nM were prepared in HBSS buffer in a final volume of 100 μL from a stock solution of 25 nM hemin (Frontier Scientific, Logan, UT, USA) and incubated for 10 min at 4°C. Then, 5 μL of each sample and standard were added to a 96-well plate. To start the assay, 200 μL of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate was added per well. To determine the concentration of labile heme, the absorbance was measured at 652 nm for 2–3 min. The time point at which the highest hemin standard induced an absorbance from 1.6 to 2 was used.
Hemin
Manufactured by Frontier Scientific
Sourced in United States
Hemin is a chemical compound that is commonly used in scientific research and laboratory applications. It is a reddish-brown crystalline solid that is derived from heme, a component of hemoglobin. Hemin serves as a cofactor for various enzymes and plays a role in various biological processes.
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Lab products found in correlation
Deferoxamine dfo, by Merck Group (2 mentions)
Brain heart infusion media, by BD (2 mentions)
Vitamin k1, by Merck Group (2 mentions)
Schaedler s broth, by BD (2 mentions)
Bovine hemoglobin, by Merck Group (2 mentions)
Mannitol, by Merck Group (1 mentions)
Superoxide dismutase, by Merck Group (1 mentions)
Sodium azide nan3, by Merck Group (1 mentions)
Glucose oxidase, by Merck Group (1 mentions)
Human fibronectin, by R&D Systems (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
1h 1 2 4 oxadiazolo 4 3 a quinoxalin 1 one, by Cayman Chemical (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Bay 60 2770, by Bayer (1 mentions)
Bay 41 2272, by Bayer (1 mentions)
Kainic acid, by Bio-Techne (1 mentions)
Paraquat, by Santa Cruz Biotechnology (1 mentions)
32 protocols using hemin
1
Labile Heme Quantification in PL Fluid
2
Porphyrin Compound Activity Assay
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The reagents we used included the following: PP-IX, Zn-PP, TMPD, ethyl sorbate, sodium azide (NaN3), mannitol, glucose oxidase, and superoxide dismutase were purchased from Sigma-Aldrich. Catalase was from Calbiochem (Burlington, MA). Hemin, Uro, and Copro were obtained from Frontier Scientific (Logan, UT). POBN was from Cayman Chemicals (Ann Arbor, MI). The following stock solutions were prepared for subsequent use: PP-IX (1.78 mmol/L in dimethylacetamide [DMA]); Hemin (1.53 mmol/L in 100 mmol/L NaOH); Zn-PP (0.5 mmol/L in DMA); Uro (5 mmol/L in 100 mmol/L NaOH); Copro (5 mmol/L in 100 mmol/L NaOH); NaN3 (100 mmol/L in 400 mmol/L phosphate buffer, pH 7.4); mannitol (100 mmol/L in 400 mmol/L phosphate buffer, pH 7.4); TMPD (121.7 mmol/L in DMA); and trolox (100 mmol/L in DMA). A detailed list of the reagents used is included in Appendix I .
3
Molecular Signaling Pathway Analysis
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BAY 60-2770 and BAY 41-2272 were provided by Bayer AG (Wuppertal, Germany). 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) was purchased from Cayman Chemical (Ann Arbor, MI), and hemin was obtained from Frontier Scientific (Newark, NJ). Recombinant human and murine tumor necrosis factor-α (TNF) and human fibronectin were from R&D Systems (Minneapolis, MN). Dulbecco’s modified Eagle’s medium, fetal bovine serum, penicillin, and streptomycin were from Gibco-Invitrogen (New York, NY). Trizol was from Invitrogen (Carlsbad, CA). All other reagents were from Sigma-Aldrich (St. Louis, MO) unless otherwise stated.
4
Oxidative Stress and Excitotoxicity Assays
5
Antibody Sources and Protein Reagents
6
Modulatory Effects of IL-10 and Compounds on OPCs
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Unless indicated specifically, we pretreated OPCs with recombinant murine IL-10 (2.5, 5, 10, 20, 40 ng/mL, 210-10, PeproTech) for 2 h. 50 μM hemin (16009-13-5, Frontier Scientific, USA), ND-646 (0.5, 5, 10 nM, HY-101842, MedChemExpress, USA), 10 μM AG490 (HY-12000, MedChemExpress), 2.5 μM Stattic (S7024, Selleck, USA) and 100 μM Deferoxamine (DFO, Y0001937, Sigma) were administrated on OPCs for 12 h.
To intracerebroventricularly administrate drugs into ICH mice, we fixed microtube (62001, RWD, China) on the skull (coordinate relative to bregma: x = 0.8 mm, y = 0.5 mm). 10 μg/kg IL-10 in ddH2O, 10 mM AG490 in 0.1% DMSO, 1 μM ND-646 (1 μL) in 0.1% DMSO and vehicle were injected at 0, 2, 4, 6 d after ICH.
To intracerebroventricularly administrate drugs into ICH mice, we fixed microtube (62001, RWD, China) on the skull (coordinate relative to bregma: x = 0.8 mm, y = 0.5 mm). 10 μg/kg IL-10 in ddH2O, 10 mM AG490 in 0.1% DMSO, 1 μM ND-646 (1 μL) in 0.1% DMSO and vehicle were injected at 0, 2, 4, 6 d after ICH.
7
Bio-Tyr Mediated Protein Labeling Protocol
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Biotin tyramide (‘bio-tyr’; Toronto Research Chemicals) was dissolved in dimethyl
sulfoxide as a 100 mM stock. Hemin or Fe(III)-heme (Frontier Scientific) stock solutions
were prepared fresh in dimethyl formamide to 1 mM. H2O2 working
solutions were diluted from a 17.4 M stock to give 100 mM in ddH2O. DNA stock
solutions were diluted in reaction buffer (final concentration: 40 mM HEPES pH 8.0, 20 mM
potassium chloride, 1% dimethylformamide, 0.05% Triton X-100) and heated to 95°C for 3
min, then cooled to room temperature. Hemin was added, followed bio-tyr and the solution
rested for 10 min prior to initiating the peroxidase reaction by addition of
H2O2 to the various final concentrations described below.
Reactions were allowed to proceed for 30 min, generally followed by quenching of the
reaction by ethanol precipitation or by addition of 10 U bovine liver catalase enzyme
(Sigma) followed by ethanol precipitation (as indicated). For the streptavidin gel shift
experiments the recovered DNA was dissolved in a streptavidin-containing water solution
for 5 min, followed by the addition of gel loading buffer. For footprinting analysis, the
samples were redissolved in 10% piperidine and heated at 90°C for 30 min, vacuum dried and
then treated with streptavidin and loading dye, as above.
sulfoxide as a 100 mM stock. Hemin or Fe(III)-heme (Frontier Scientific) stock solutions
were prepared fresh in dimethyl formamide to 1 mM. H2O2 working
solutions were diluted from a 17.4 M stock to give 100 mM in ddH2O. DNA stock
solutions were diluted in reaction buffer (final concentration: 40 mM HEPES pH 8.0, 20 mM
potassium chloride, 1% dimethylformamide, 0.05% Triton X-100) and heated to 95°C for 3
min, then cooled to room temperature. Hemin was added, followed bio-tyr and the solution
rested for 10 min prior to initiating the peroxidase reaction by addition of
H2O2 to the various final concentrations described below.
Reactions were allowed to proceed for 30 min, generally followed by quenching of the
reaction by ethanol precipitation or by addition of 10 U bovine liver catalase enzyme
(Sigma) followed by ethanol precipitation (as indicated). For the streptavidin gel shift
experiments the recovered DNA was dissolved in a streptavidin-containing water solution
for 5 min, followed by the addition of gel loading buffer. For footprinting analysis, the
samples were redissolved in 10% piperidine and heated at 90°C for 30 min, vacuum dried and
then treated with streptavidin and loading dye, as above.
8
Preparation of Heme-Albumin Solution
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The term “heme” is used generically to refer to both heme and hemin. These studies used hemin that was prepared immediately before use either dissolved only in NaOH or as heme-albumin. Briefly, 65 mg hemin (Frontier Scientific, Logan, UT, USA) was dissolved in 10 mL NaOH (0.1 M) at 37 °C, and then 15 mL of 20% human serum albumin (CSL Behring AG, Bern, Switzerland) was added. After 1 h of incubation at 37 °C, the pH of the solution was adjusted to pH 7.4–7.8 using 85% orthophosphoric acid. The 4 mmol/L heme-albumin solution was sterile-filtered (0.22 µm) and used immediately.
9
Hemin-Induced THP-1 Cell Treatment
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Hemin was obtained from Frontier Scientific. Blood samples were collected from healthy control subjects. RBCs were isolated by centrifugation, frozen at −80°C for 15 min, and then thawed for lysis. Cellular debris was pelleted by centrifugation, and the hemolysates were stored at −80°C. Differentiated THP-1 cells were treated either with different concentrations of Hemin or hemolysates for 18 h.
10
Culturing Anaerobic Gut Bacteria
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