Light-induced permeabilization and merocyanine 540 staining of mouse spleen cells

Szabó, Gábor; Rédai, Imre; Bacsó, Zsolt; Hovessy, Jozsef; Damjanovich, Sándor

doi:10.1016/0005-2736(89)90258-7

Cited by 9 publications

(4 citation statements)

References 26 publications

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“…Several groups have reported evidence that PS exposure in live sperm cells represents an early sign of cellular damage (Glander and Schaller, 1999;Ramos and Wetzels, 2001;Muratori et al, 2003Muratori et al, , 2004). An increase in merocyanine fluorescence has also been attributed to an increase in membrane permeability, possibly related to penetration of merocyanine to the more fluid inner membrane structures (Falke and Lazarides, 1980;Szabo et al, 1989;Berthiaume and Frangos, 1994) In this study, dead spermatozoa exhibited high merocyanine fluorescence, which was no doubt related to the loss of membrane integrity. Merocyanine has also been used as an marker of apoptosis (Reid et al, 1996;Laakko et al, 2002) and as an indicator of membrane potential (Waggoner, 1979).…”

Section: Discussionmentioning

confidence: 56%

Changes in Membrane Lipid Order With Capacitation in Rhesus Macaque (Macaca mulatta) Spermatozoa

Baumber

Meyers

2006

Journal of Andrology

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Lipophilic fluorescent dye merocyanine 540 is believed to stain cell membranes with increasing affinity as the lipid components become more disordered and has been associated with changes in membrane fluidity. The aim of this study was to determine whether membrane lipid disorder is associated with capacitation of macaque spermatozoa. To induce capacitation, spermatozoa from 5 rhesus macaques were incubated at 37uC (5% CO 2 in air) for 2 hours in a modified Biggers-WhittenWhittingham medium containing 30 mg/mL bovine serum albumin and 36 mmol/L NaHCO 3 . Caffeine (1 mmol/L) and dbcAMP (1.2 mmol/L) were added to the medium, and incubation was performed for an additional 30 minutes. Sperm motility was determined by computer-assisted sperm analysis, and membrane lipid order and sperm viability was determined by flow cytometry with merocyanine (2.7 mmol/L) and Yo-Pro-1 (25 nmol/L), respectively. Tyrosine phosphorylation of proteins in sperm tail was immunohistochemically examined by means of anti-phosphotyrosine (a-PY; clone 4G10). Capacitation resulted in a significant increase in the amplitude of lateral head displacement and beat cross frequency (P , .005) and a significant decrease in linearity and straightness in capacitated spermatozoa (P , .005), compared with control spermatozoa, which suggests the expression of hyperactivated motility. Animals in which capacitation was induced had a significant increase in the number of spermatozoa showing tyrosine phosphorylation of tail proteins (P , .0001) and a significant increase in the intensity of merocyanine fluorescence (P , .0001), compared with control animals. The observed decrease in membrane lipid order after capacitation was induced was not associated with surface exposure of phosphatidylserine, as determined by flow cytometry with annexin V-Alexa Fluor 488. Merocyanine may be a useful tool for investigating the role of the plasma membrane on capacitation and other cytotoxic events in macaque spermatozoa.

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Section: Discussionmentioning

confidence: 56%

Changes in Membrane Lipid Order With Capacitation in Rhesus Macaque (Macaca mulatta) Spermatozoa

Baumber

Meyers

2006

Journal of Andrology

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“…It seems likely that other cell types may be similarly affected. For instance, Szabo Jr. et al have shown that the plasma membrane of mouse spleen cells becomes permeable to MC540 upon oxidative stress induced by UV irradiation, thereby allowing the dye to bind to intracellular structures [26]. In previous studies it was concluded that the increased phagocytosis of H 2 O 2 -treated erythrocytes by macrophages was due to a looser packing of the phospholipids in the outer leaflet of the erythrocyte membrane.…”

Section: Discussionmentioning

confidence: 99%

Effect of hydrogen peroxide on the binding of Merocyanine 540 to human erythrocytes

Lagerberg

VanSteveninck

Dubbelman

1997

CMLS, Cell. mol. life sci.

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The fluorescent dye Merocyanine 540 (MC540) is often used as a probe to monitor the molecular packing of phospholipids in the outer leaflet of biomembranes. In a previous study we showed that the increased staining of erythrocytes with a perturbed membrane structure was mainly due to an increase in the fluorescence yield of cell-bound MC540, rather than to an increase of the number of bound molecules. Erythrocytes and ghosts exposed to continuous fluxes of H2O2 exhibited pronounced lipid peroxidation. Further, red blood cells subjected to this form of oxidative stress also showed increased staining with MC540. It appeared that this was caused by a strong increase in binding of MC540, together with a slight red shift of the fluorescence emission maximum and a small increase in the fluorescence yield of bound MC540. The changed MC540 binding characteristics were not observed when lipid peroxidation was suppressed by the presence of the antioxidant BHT in the incubation medium. However, open ghosts exposed to H2O2 showed no increase of MC540 binding, excluding a direct involvement of lipid peroxidation. Measurement of fluorescence emission spectra and gel filtration studies showed that MC540 can bind to H2O2-exposed hemoglobin. Experiments with erythrocytes lysed in hypotonic medium after exposure to H2O2 revealed that peroxidation of lipids with H2O2 induced a non-specific permeabilization of the plasma membrane to MC540, thereby allowing MC540 to bind to the oxidatively denatured, more hydrophobic hemoglobin. These results indicate that conclusions about packing of phospholipids in the outer leaflet of the membrane based on increased MC540-staining should be drawn with care.

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“…These results showed that increase of NBD-PS translocation oc-curred because of small doses of UVA that were not lethal. Oxidation by UV irradiation is known to increase membrane permeability in both hydrophobic and hydrophilic molecules (26)(27)(28), which was considered to cause the observed increase in NBD-PS uptake into the inner leaflet. The cells were stained with both NBD-PS and PI and the uptake of NBD-PS into the inner leaflet in PI-unstained cells was determined by flow cytometry (Fig.…”

Section: Methodsmentioning

confidence: 99%

UVA Irradiation Induces Energy-independent Phospholipid-flip in Mammalian Plasma Membrane¶

Ibuki¹,

Suzuki²,

Gotô³

2007

Photochemistry and Photobiology

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Translocation from the outer to the inner membrane leaflet (flip) of phospholipids after ultraviolet A (UVA) irradiation was investigated in Chinese hamster ovary cells. Fluorescent 1‐palmitoyl‐2‐[6‐[(7‐nitro‐2‐1,3‐benzoxadiazol‐4‐yl)amino]caproyl]‐sn‐glycero‐3‐phosphoserine (NBD‐labeled phosphatidylserine [NBD‐PS]) was used to assay transbilayer lipid movement. A marked increase in flip of NBD‐PS was observed immediately after low‐dose UVA irradiation which was not lethal and returned to the basal level after 6 h. UVA‐induced flip was not attributed to the increase of permeability by UVA irradiation because cells that were negative for staining with propidium iodide also showed increased flip of NBD‐PS. Furthermore, the enhancement was independent of adenosine 5′‐triphosphate, demonstrating the lack of involvement of phospholipid translocase. Marked increases were also observed in flip of both NBD‐phosphatidylethanolamine and NBD‐phosphatidylcholine immediately after UVA irradiation, showing that the increase was independent on the head groups of phospholipids. These findings indicated that UVA changes the flip–flop of phospholipids and that the cell membrane is a molecular and cellular target of UVA.

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Light-induced permeabilization and merocyanine 540 staining of mouse spleen cells

Cited by 9 publications

References 26 publications

Changes in Membrane Lipid Order With Capacitation in Rhesus Macaque (Macaca mulatta) Spermatozoa

Changes in Membrane Lipid Order With Capacitation in Rhesus Macaque (Macaca mulatta) Spermatozoa

Effect of hydrogen peroxide on the binding of Merocyanine 540 to human erythrocytes

UVA Irradiation Induces Energy-independent Phospholipid-flip in Mammalian Plasma Membrane¶

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