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Methods Employed in Cytofluorometric Assessment of Eryptosis, the Suicidal Erythrocyte Death

Jemaà, Mohamed LU ; Fezai, Myriam; Bissinger, Rosi and Lang, Florian (2017) In Cellular Physiology and Biochemistry 43(2). p.431-444
Abstract

Suicidal erythrocyte death or eryptosis contributes to or even accounts for anemia in a wide variety of clinical conditions, such as iron deficiency, dehydration, hyperphosphatemia, vitamin D excess, chronic kidney disease (CKD), hemolytic-uremic syndrome, diabetes, hepatic failure, malignancy, arteriitis, sepsis, fever, malaria, sickle-cell disease, beta-thalassemia, Hb-C and G6PD-deficiency, Wilsons disease, as well as advanced age. Moreover, eryptosis is triggered by a myriad of xenobiotics and endogenous substances including cytotoxic drugs and uremic toxins. Eryptosis is characterized by cell membrane scrambling with phosphatidylserine exposure to the erythrocyte surface. Triggers of eryptosis include oxidative stress, hyperosmotic... (More)

Suicidal erythrocyte death or eryptosis contributes to or even accounts for anemia in a wide variety of clinical conditions, such as iron deficiency, dehydration, hyperphosphatemia, vitamin D excess, chronic kidney disease (CKD), hemolytic-uremic syndrome, diabetes, hepatic failure, malignancy, arteriitis, sepsis, fever, malaria, sickle-cell disease, beta-thalassemia, Hb-C and G6PD-deficiency, Wilsons disease, as well as advanced age. Moreover, eryptosis is triggered by a myriad of xenobiotics and endogenous substances including cytotoxic drugs and uremic toxins. Eryptosis is characterized by cell membrane scrambling with phosphatidylserine exposure to the erythrocyte surface. Triggers of eryptosis include oxidative stress, hyperosmotic shock, and energy depletion. Signalling involved in the regulation of eryptosis includes Ca2+ entry, ceramide, caspases, calpain, p38 kinase, protein kinase C, Janus-activated kinase 3, casein kinase 1α, cyclin-dependent kinase 4, AMP-activated kinase, p21-activated kinase 2, cGMP-dependent protein kinase, mitogen- and stress-activated kinase MSK1/2, and ill-defined tyrosine kinases. Inhibitors of eryptosis may prevent anaemia in clinical conditions associated with enhanced eryptosis and stimulators of eryptosis may favourably influence the clinical course of malaria. Additional experimentation is required to uncover further clinical conditions with enhanced eryptosis, as well as further signalling pathways, further stimulators, and further inhibitors of eryptosis. Thus, a detailed description of the methods employed in the analysis of eryptosis may help those, who enter this exciting research area. The present synopsis describes the experimental procedures required for the analysis of phosphatidylserine exposure at the cell surface with annexin-V, cell volume with forward scatter, cytosolic Ca2+ activity ([Ca2+]) with Fluo3, oxidative stress with 2′,7′-dichlorodihydrofuorescein diacetate (DCFDA), glutathione (GSH) with mercury orange 1(4-chloromercuryphenyl-azo-2-naphthol), lipid peroxidation with BODIPY 581/591 C11 fluorescence, and ceramide abundance with specific antibodies. The contribution of kinases and caspases is defined with the use of the respective inhibitors. It is hoped that the present detailed description of materials and methods required for the analysis of eryptosis encourages further scientists to enter this highly relevant research area.

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organization
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Contribution to journal
publication status
published
subject
keywords
Bi-2536, Calcium, Caspases, Cell membrane scrambling, Cell volume, Ceramide, Fascaplycin, Fucoxanthin, Glutathion, Kinases, Lopinavir, NSC-95397, RBC, ROS, Terfenadine
in
Cellular Physiology and Biochemistry
volume
43
issue
2
pages
14 pages
publisher
Karger
external identifiers
  • scopus:85032492269
  • wos:000415241500001
ISSN
1015-8987
DOI
10.1159/000480469
language
English
LU publication?
yes
id
30b2ac6c-d73b-4fc2-bf1f-2b0a55f87994
date added to LUP
2017-11-07 14:17:07
date last changed
2018-05-20 04:38:36
@article{30b2ac6c-d73b-4fc2-bf1f-2b0a55f87994,
  abstract     = {<p>Suicidal erythrocyte death or eryptosis contributes to or even accounts for anemia in a wide variety of clinical conditions, such as iron deficiency, dehydration, hyperphosphatemia, vitamin D excess, chronic kidney disease (CKD), hemolytic-uremic syndrome, diabetes, hepatic failure, malignancy, arteriitis, sepsis, fever, malaria, sickle-cell disease, beta-thalassemia, Hb-C and G6PD-deficiency, Wilsons disease, as well as advanced age. Moreover, eryptosis is triggered by a myriad of xenobiotics and endogenous substances including cytotoxic drugs and uremic toxins. Eryptosis is characterized by cell membrane scrambling with phosphatidylserine exposure to the erythrocyte surface. Triggers of eryptosis include oxidative stress, hyperosmotic shock, and energy depletion. Signalling involved in the regulation of eryptosis includes Ca<sup>2+</sup> entry, ceramide, caspases, calpain, p38 kinase, protein kinase C, Janus-activated kinase 3, casein kinase 1α, cyclin-dependent kinase 4, AMP-activated kinase, p21-activated kinase 2, cGMP-dependent protein kinase, mitogen- and stress-activated kinase MSK1/2, and ill-defined tyrosine kinases. Inhibitors of eryptosis may prevent anaemia in clinical conditions associated with enhanced eryptosis and stimulators of eryptosis may favourably influence the clinical course of malaria. Additional experimentation is required to uncover further clinical conditions with enhanced eryptosis, as well as further signalling pathways, further stimulators, and further inhibitors of eryptosis. Thus, a detailed description of the methods employed in the analysis of eryptosis may help those, who enter this exciting research area. The present synopsis describes the experimental procedures required for the analysis of phosphatidylserine exposure at the cell surface with annexin-V, cell volume with forward scatter, cytosolic Ca<sup>2+</sup> activity ([Ca<sup>2+</sup>]) with Fluo3, oxidative stress with 2′,7′-dichlorodihydrofuorescein diacetate (DCFDA), glutathione (GSH) with mercury orange 1(4-chloromercuryphenyl-azo-2-naphthol), lipid peroxidation with BODIPY 581/591 C11 fluorescence, and ceramide abundance with specific antibodies. The contribution of kinases and caspases is defined with the use of the respective inhibitors. It is hoped that the present detailed description of materials and methods required for the analysis of eryptosis encourages further scientists to enter this highly relevant research area.</p>},
  author       = {Jemaà, Mohamed and Fezai, Myriam and Bissinger, Rosi and Lang, Florian},
  issn         = {1015-8987},
  keyword      = {Bi-2536,Calcium,Caspases,Cell membrane scrambling,Cell volume,Ceramide,Fascaplycin,Fucoxanthin,Glutathion,Kinases,Lopinavir,NSC-95397,RBC,ROS,Terfenadine},
  language     = {eng},
  month        = {10},
  number       = {2},
  pages        = {431--444},
  publisher    = {Karger},
  series       = {Cellular Physiology and Biochemistry},
  title        = {Methods Employed in Cytofluorometric Assessment of Eryptosis, the Suicidal Erythrocyte Death},
  url          = {http://dx.doi.org/10.1159/000480469},
  volume       = {43},
  year         = {2017},
}