Flow cytometry measurement of the labile iron pool in human hematopoietic cells

Prus, Eugenia; Fibach, Eitan

doi:10.1002/cyto.a.20491

Cited by 114 publications

(111 citation statements)

References 26 publications

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“…The level of the LIP is regulated and maintained within a restricted range that meets the cell's requirements for iron but prevents excess free iron from triggering cellular damage. [36][37][38][39] As shown in Figure 6a, compared with the control, the level of the LIP was upregulated after cells were treated with c-leachate, whereas treatment with CNT-2 had little effect on levels of the LIP. Upregulation of the LIP indicates that intracellular iron is more abundant and therefore could be available for ironcatalyzed reactions generating ROS and resulting in oxidative stress.…”

Section: Measurement Of Intracellular Transferrin Receptor (Tfr) Exprmentioning

confidence: 87%

The contributions of metal impurities and tube structure to the toxicity of carbon nanotube materials

et al. 2012

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Due to the existence of considerable quantities of metallic and carbonaceous impurities, the key factor and mechanism for the reported toxicity of carbon nanotubes (CNTs) are unclear. Here, we first quantify the contribution of metal residues and fiber structure to the toxicity of CNTs. Significant quantities of metal particles could be mobilized from CNTs into surrounding fluids, depending on the properties and constituents of the biological microenvironment, as well as the properties of metal particles. Furthermore, electron spin resonance measurements confirm that hydroxyl radicals can be generated by both CNTs containing metal impurities and acid-leachable metals from CNTs. Several biomolecules facilitate the generation of free radicals, which might be due to the participation of these biomolecules in redox cycling influenced by pH. Among several major metal residues, Fe has a critical role in generating hydroxyl radicals, reducing cell viability and promoting intracellular reactive oxidative species. Cell viability is highly dependent on the amount of metal residues and iron in particular, but not tube structure, while the negative effect of CNTs themselves on cell viability is very limited in a certain concentration range below 80 lg ml À1 . It is crucial to systematically understand how these exogenous and endogenous factors influence the toxicity of CNTs to avoid their undesirable toxicity. Keywords: biological microenvironments; carbon nanotube; metal impurities; reactive oxygen species; toxicity INTRODUCTION Engineered nanomaterials possess novel properties and have been used worldwide in numerous consumer products, for example, food, clothes, pharmaceuticals and cleaning products. Estimates suggest that by 2015 nanotechnology will have a trillion-plus dollar global economic impact. 1 Therefore, ensuring the safety of nanomaterials is of great importance to the tremendous commercial applications of nanotechnology. Safety evaluation of nanomaterials should consider their behaviors in various aspects, including their interaction with proteins, DNA, lipids, membranes, organelles, cells, tissues, biological fluids and even the dissolution of metal constituents. [2][3][4] Carbon nanotubes (CNTs) have attracted great interest from both scientists and the industry because their high aspect ratios, strength and remarkable physical properties make them a unique material with a wide range of promising applications. Many applications of CNTs in biomedicine have been proposed, including nanoelectronics, 4 biosensors, 5 biomolecular recognition devices and molecular transporters, 6 artificial water channels, 7 cancer therapy 8,9 and photoacoustic molecular imaging. 10 Because transitional metals are often used as catalysts during CNT synthesis, 11 it is unavoidable that as-received CNTs are contaminated by catalyst residues. 12 The catalyst precursor (such as iron carbonyl) decomposes, and nanometer-sized metal particles form from the decomposition products. Therefore,

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Section: Measurement Of Intracellular Transferrin Receptor (Tfr) Exprmentioning

confidence: 87%

The contributions of metal impurities and tube structure to the toxicity of carbon nanotube materials

et al. 2012

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“…LIP was measured according to the methods described by Prus and Fibach [35]. Briefly, cells were trypsinized, washed twice with 0.5 ml of PBS, and incubated at a density of 1 × 10 6 /ml for 15 min at 37 °C with 0.05 µM calcein-acetoxymethyl ester (AnaSpec).…”

Section: Measurement Of Lipmentioning

confidence: 99%

Ferroptosis is an autophagic cell death process

et al. 2016

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Ferroptosis is an iron-dependent form of regulated necrosis. It is implicated in various human diseases, including ischemic organ damage and cancer. Here, we report the crucial role of autophagy, particularly autophagic degradation of cellular iron storage proteins (a process known as ferritinophagy), in ferroptosis. Using RNAi screening coupled with subsequent genetic analysis, we identified multiple autophagy-related genes as positive regulators of ferroptosis. Ferroptosis induction led to autophagy activation and consequent degradation of ferritin and ferritinophagy cargo receptor NCOA4. Consistently, inhibition of ferritinophagy by blockage of autophagy or knockdown of NCOA4 abrogated the accumulation of ferroptosis-associated cellular labile iron and reactive oxygen species, as well as eventual ferroptotic cell death. Therefore, ferroptosis is an autophagic cell death process, and NCOA4-mediated ferritinophagy supports ferroptosis by controlling cellular iron homeostasis.

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“…CA fluorescence was measured at 530 nm, using the FL1 photomultiplier tube with logarithmic amplification. 25,26 Cells were analyzed on a BD flow cytometer (BD Biosciences, San Jose, CA), and raw files were analyzed by FlowJo (TreeStar Inc., Ashland, OR). LIP was determined based on the ability of chelators (e.g., deferiprone [L1]) to remove LIP bound to fluorescent CA-AM.…”

Section: Multiparameter Flow Cytometry For Measuring Lipmentioning

confidence: 99%

“…LIP was determined based on the ability of chelators (e.g., deferiprone [L1]) to remove LIP bound to fluorescent CA-AM. 25,26 Iron-Regulatory Gene Expression in Liver in Patients With ACLF and Cirrhosis…”

Section: Multiparameter Flow Cytometry For Measuring Lipmentioning

confidence: 99%

Dysregulated iron homeostasis is strongly associated with multiorgan failure and early mortality in acute‐on‐chronic liver failure

et al. 2015

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Acute-on-chronic liver failure (ACLF) is an ailment with high incidence of multiorgan failure (MOF) and consequent mortality. Dysregulated iron homeostasis and macrophage dysfunction are linked to increased incidence of MOF. We investigated whether a panel of circulating iron-regulating proteins are associated with development of MOF and can predict 15-or 30-day mortality in ACLF patients. One hundred twenty patients with ACLF, 20 patients with compensated cirrhosis, and 20 healthy controls were studied. Relative protein expression profiling was performed in the derivative cohort and confirmed in the validation cohort. A panel of iron regulators and indices were determined. Multiparametric flow cytometry for quantitation of labile iron pool (LIP) was performed. Validation studies confirmed lower serum transferrin (Tf) and ceruloplasmin levels in ACLF and ACLF-MOF, compared to patients with cirrhosis and controls (P < 0.01). Serum iron and ferritin levels were markedly elevated (P < 0.001; P < 0.05) and hepcidin levels were lower (P < 0.001) in ACLF patients with MOF than those without and other groups (P < 0.001). Percentage Tf saturation (%SAT) was higher in ACLF-MOF (39.2%; P < 0.001) and correlated with poor outcome (hazard ratio: 6.970; P < 0.01). Intracellular LIP indices were significantly elevated in the subsets of circulating macrophages in ACLF-MOF, compared to other groups (P < 0.01). Whereas expression of iron-regulatory genes was markedly down-regulated, genes related to endoplasmic reticulum stress, apoptosis, and inflammation were up-regulated in ACLF patients, compared to patients with cirrhosis. Severe dysregulation of autophagy mechanisms was also observed in the former. Conclusions: Iron metabolism and transport are severely deranged in ACLF patients and more so in those with MOF. %SAT, circulating hepcidin, and LIP in macrophages correlate with disease severity and %SAT could be used for early prognostication in ACLF patients. (HEPATOLOGY 2015;61:1306-1320

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Flow cytometry measurement of the labile iron pool in human hematopoietic cells

Cited by 114 publications

References 26 publications

The contributions of metal impurities and tube structure to the toxicity of carbon nanotube materials

The contributions of metal impurities and tube structure to the toxicity of carbon nanotube materials

Ferroptosis is an autophagic cell death process

Dysregulated iron homeostasis is strongly associated with multiorgan failure and early mortality in acute‐on‐chronic liver failure

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