Neuroprotective Agents against Hemin-Induced Cell Death

Blood breakdown product, hemin, was used to induce cell death in primary cortical neurons. For the protective studies, cells were treated with hemin (100 μM) in the presence of NAC (0.01–1.00mM), Trolox (0.1–100.0 μM), α‐lipoic acid (0.01–2.00mM), U 73122 (0.1–100.0 μM), β carotene (0.1–100.0 μM), MS‐PPOH (0.1–100.0 μM), aspirin (0.1–100.0 μM), celecoxib (0.1–100.0 μM), Indomethacin (0.1–100.0 μM), Zileuton (0.1–100.0 μM), BW B70 (0.1–100.0 μM), BW A4C (0.1–100.0 μM), NCTT‐956 (0.1–100.0 μM), PD146176 (0.1–100.0 μM), MK 561 (0.1–100.0 μM), glutathione ethyl ester (1–10mM), L‐oxothiazolidine‐4‐carboxylate (1–10mM), cystamine (0.1–10.0 μM), and nordihydroguaiaretic acid (0.1–10.0 μM). Cell viability was analyzed 24 hours after treatment. Cells were rinsed with warm phosphate‐buffered saline (PBS) and assessed by methyl thiazolyl tetrazolium (MTT) assay. The fidelity of MTT assays in measuring viability was verified by calcein‐AM/ethidium homodimer‐1 staining (Live/Dead assay; Molecular Probes, Eugene, OR), following the manufacturer's instructions.

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Karuppagounder S.S., Alin L., Chen Y., Brand D., Bourassa M.W., Dietrich K., Wilkinson C.M., Nadeau C.A., Kumar A., Perry S., Pinto J.T., Darley‐Usmar V., Sanchez S., Milne G.L., Pratico D., Holman T.R., Carmichael S.T., Coppola G., Colbourne F, & Ratan R.R. (2018). N‐acetylcysteine targets 5 lipoxygenase‐derived, toxic lipids and can synergize with prostaglandin E2 to inhibit ferroptosis and improve outcomes following hemorrhagic stroke in mice. Annals of Neurology, 84(6), 854-872.

Publication 2018

Live/Dead assay

After treatment Aspirin Assay Blood Breakdown Bw a4c Calcein Carotene Celecoxib Cell death Cell viability Cells Cortical Cystamine Ethidium homodimer 1 Glutathione ethyl ester Hemin Indomethacin Molecular probes Ms ppoh Neurons Nordihydroguaiaretic acid Phosphate Saline Tetrazolium Trolox U 73122 Zileuton α lipoic acid

Corresponding Organization : Cornell University

Other organizations : Women and Children’s Health Research Institute, University of Alberta, University of California, Santa Cruz, New York Medical College, University of Alabama at Birmingham, Vanderbilt University, Temple University, University of California, Los Angeles

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Variable analysis

independent variables

Hemin (100 μM)
NAC (0.01–1.00mM)
Trolox (0.1–100.0 μM)
α‐lipoic acid (0.01–2.00mM)
U 73122 (0.1–100.0 μM)
β carotene (0.1–100.0 μM)
MS‐PPOH (0.1–100.0 μM)
Aspirin (0.1–100.0 μM)
Celecoxib (0.1–100.0 μM)
Indomethacin (0.1–100.0 μM)
Zileuton (0.1–100.0 μM)
BW B70 (0.1–100.0 μM)
BW A4C (0.1–100.0 μM)
NCTT‐956 (0.1–100.0 μM)
PD146176 (0.1–100.0 μM)
MK 561 (0.1–100.0 μM)
Glutathione ethyl ester (1–10mM)
L‐oxothiazolidine‐4‐carboxylate (1–10mM)
Cystamine (0.1–10.0 μM)
Nordihydroguaiaretic acid (0.1–10.0 μM)

dependent variables

Cell viability

control variables

Primary cortical neurons

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