A novel cytotoxicity screening assay using a multiwell fluorescence scanner

Nieminen, Anna‐Liisa; Gores, Gregory J.; Bond, John M.; Imberti, Roberto; Herman, Brian; Lemasters, John J.

doi:10.1016/0041-008x(92)90317-l

Cited by 122 publications

(78 citation statements)

References 35 publications

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“…At the end of the experiment, digitonin (200 M) was added to each well to permeabilize all cells and label all nuclei with PI. Cell viability determined by PI fluorometry is essentially the same as cell viability determined by trypan blue exclusion (27).…”

Section: Methodsmentioning

confidence: 91%

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Mitoferrin-2-dependent Mitochondrial Iron Uptake Sensitizes Human Head and Neck Squamous Carcinoma Cells to Photodynamic Therapy

Hung

Schwartz²,

Maldonado

et al. 2013

Journal of Biological Chemistry

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Background: Here, we investigated whether iron mobilization enhanced photodynamic therapy (PDT)-induced cell killing. Results: Lysosomal iron release and mitochondrial iron uptake through mitoferrin-2 (Mfrn2) acted synergistically to induce PDT-mediated and iron-dependent mitochondrial dysfunction and cell killing. Conclusion: Mfrn2 plays a critical role in transporting iron to mitochondria to enhance PDT. Significance: Head and neck cancers expressing higher Mfrn2 protein levels benefit more from PDT.

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Section: Methodsmentioning

confidence: 91%

“…Assessment of Cell Death-Cell death was assessed by propidium iodide (PI) fluorometry using a multiwell fluorescence plate reader as described previously (27). PI fluorescence was measured at frequent intervals using 530-nm excitation (25-nm band pass) and 620-nm emission (40-nm band pass) filters.…”

Section: Methodsmentioning

confidence: 99%

Mitoferrin-2-dependent Mitochondrial Iron Uptake Sensitizes Human Head and Neck Squamous Carcinoma Cells to Photodynamic Therapy

Hung

Schwartz²,

Maldonado

et al. 2013

Journal of Biological Chemistry

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“…Necrosis was assessed by a PI fluorometry using a multiwell fluorescence scanner (SpectraMax M2; Molecular device, Sunnyvale, CA, USA), as previously described (Kim, Qian & Lemasters, 2003; Nieminen et al., 1992). Intracellular levels of NAD + and NADH were colorimetrically determined with a commercial kit (AAT Bioquest, Sunnyvale, CA, USA).…”

Section: Methodsmentioning

confidence: 99%

Loss of sirtuin 1 and mitofusin 2 contributes to enhanced ischemia/reperfusion injury in aged livers

et al. 2018

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SummaryIschemia/reperfusion (I/R) injury is a causative factor contributing to morbidity and mortality during liver resection and transplantation. Livers from elderly patients have a poorer recovery from these surgeries, indicating reduced reparative capacity with aging. Mechanisms underlying this age‐mediated hypersensitivity to I/R injury remain poorly understood. Here, we investigated how sirtuin 1 (SIRT1) and mitofusin 2 (MFN2) are affected by I/R in aged livers. Young (3 months) and old (23–26 months) male C57/BL6 mice were subjected to hepatic I/R in vivo. Primary hepatocytes isolated from each age group were also exposed to simulated in vitro I/R. Biochemical, genetic, and imaging analyses were performed to assess cell death, autophagy flux, mitophagy, and mitochondrial function. Compared to young mice, old livers showed accelerated liver injury following mild I/R. Reperfusion of old hepatocytes also showed necrosis, accompanied with defective autophagy, onset of the mitochondrial permeability transition, and mitochondrial dysfunction. Biochemical analysis indicated a near‐complete loss of both SIRT1 and MFN2 after I/R in old hepatocytes, which did not occur in young cells. Overexpression of either SIRT1 or MFN2 alone in old hepatocytes failed to mitigate I/R injury, while co‐overexpression of both proteins promoted autophagy and prevented mitochondrial dysfunction and cell death after reperfusion. Genetic approaches with deletion and point mutants revealed that SIRT1 deacetylated K655 and K662 residues in the C‐terminus of MFN2, leading to autophagy activation. The SIRT1‐MFN2 axis is pivotal during I/R recovery and may be a novel therapeutic target to reduce I/R injury in aged livers.

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“…Fluorescence was measured using a multiwell fluorescence reader (BMG Lab Technologies, Offenburg, Germany), as described previously. 23 Cell killing assessed by PI fluorometry correlates closely with trypan blue exclusion and enzyme release as indicators of oncotic necrosis.…”

Section: Methodsmentioning

confidence: 99%

Mitochondrial permeability transition in acetaminophen-induced necrosis and apoptosis of cultured mouse hepatocytes

et al. 2004

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Acetaminophen overdose causes massive hepatic failure via mechanisms involving glutathione depletion, oxidative stress, and mitochondrial dysfunction. The ultimate target of acetaminophen causing cell death remains uncertain, and the role of apoptosis in acetaminophen-induced cell killing is still controversial. Our aim was to evaluate the mitochondrial permeability transition (MPT) as a key factor in acetaminophen-induced necrotic and apoptotic killing of primary cultured mouse hepatocytes. After administration of 10 mmol/L acetaminophen, necrotic killing increased to more than 49% and 74%, respectively, after 6 and 16 hours. MPT inhibitors, cyclosporin A (CsA), and NIM811 temporarily decreased necrotic killing after 6 hours to 26%, but cytoprotection was lost after 16 hours. Confocal microscopy revealed mitochondrial depolarization and inner membrane permeabilization approximately 4.5 hours after acetaminophen administration. CsA delayed these changes, indicative of the MPT, to approximately 11 hours after acetaminophen administration. Apoptosis indicated by nuclear changes, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, and caspase-3 activation also increased after acetaminophen administration. Fructose (20 mmol/L, an adenosine triphosphategenerating glycolytic substrate) plus glycine (5 mmol/L, a membrane stabilizing amino acid) prevented nearly all necrotic cell killing but paradoxically increased apoptosis from 37% to 59% after 16 hours. In the presence of fructose plus glycine, CsA decreased apoptosis and delayed but did not prevent the MPT. In conclusion, after acetaminophen a CsA-sensitive MPT occurred after 3 to 6 hours followed by a CsA-insensitive MPT 9 to 16 hours after acetaminophen. The MPT then induces ATP depletion-dependent necrosis or caspase-dependent apoptosis as determined, in part, by ATP availability from glycolysis.

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A novel cytotoxicity screening assay using a multiwell fluorescence scanner

Cited by 122 publications

References 35 publications

Mitoferrin-2-dependent Mitochondrial Iron Uptake Sensitizes Human Head and Neck Squamous Carcinoma Cells to Photodynamic Therapy

Mitoferrin-2-dependent Mitochondrial Iron Uptake Sensitizes Human Head and Neck Squamous Carcinoma Cells to Photodynamic Therapy

Loss of sirtuin 1 and mitofusin 2 contributes to enhanced ischemia/reperfusion injury in aged livers

Mitochondrial permeability transition in acetaminophen-induced necrosis and apoptosis of cultured mouse hepatocytes

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