A procedure to prepare cultured cells in suspension for electron probe X‐ray microanalysis: application to scanning and transmission electron microscopy

Fernández-Segura, None; Cañizares,; Cubero,; Campos, Antonìo; Warley,

doi:10.1046/j.1365-2818.1999.00588.x

Cited by 11 publications

(10 citation statements)

References 21 publications

(37 reference statements)

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“…Preparation of whole cells for X‐ray microanalysis was described in detail elsewhere (Fernández‐Segura et al, 1999a). Briefly, cells were removed from the culture medium and transferred into polycarbonate tissue culture plate well inserts (Millicell‐PCF), which were transferred to conical centrifuge tubes and centrifuged at 170 g for 3–5 min.…”

Section: Methodsmentioning

confidence: 99%

Changes in intracellular sodium, chlorine, and potassium concentrations in staurosporine‐induced apoptosis

Arrebola

Zabiti

Cañizares

et al. 2005

Journal Cellular Physiology

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Ion gradients across the plasma membrane, fundamentally K(+), play a pivotal role in the execution phase of apoptosis. However, little is known about other monovalent anions (Cl(-)) or cations (Na(+)) in apoptosis. In addition, the relationship between changes in total ion composition and morphological and biochemical events are poorly understood. We investigated simultaneous changes in sodium (Na), chlorine (Cl), and potassium (K) concentrations in stauroporine-induced apoptosis by quantitative electron probe X-ray microanalysis (EPXMA) in single cells. Apoptotic cells identified unequivocally from the presence of chromatin condensation in backscattered electron images were characterized by an increase in intracellular Na, a decrease in intracellular Cl and K concentrations, and a decrease in K/Na ratio. The ouabain-sensitive Rb-uptake assay demonstrated a net decrease in Na(+)/K(+)-ATPase activity, suggesting that increases in Na and decreases in K and the K/Na ratio in apoptotic cells were related with inhibition of the Na(+)/K(+)-ATPase pump. These changes in diffusible elements were associated with externalization of phosphatidyl serine and oligonucleosomal fragmentation of DNA. This alteration in ion homeostasis and morphological hallmarks of apoptosis occur in cells that have lost their inner mitochondrial transmembrane potential and before the plasma membrane becomes permeable.

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Section: Methodsmentioning

confidence: 99%

Changes in intracellular sodium, chlorine, and potassium concentrations in staurosporine‐induced apoptosis

Arrebola

Zabiti

Cañizares

et al. 2005

Journal Cellular Physiology

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“…27 Briefly, cells were removed from the culture medium and transferred into polycarbonate tissue culture plate well inserts (Millipore, Bedford, MA), which were in turn transferred to conical centrifuge tubes and centrifuged at 170×g for 3-5 min. After centrifugation, polycarbonate membrane filters were cut from their polystyrene holder and washed with ice-cold distilled water for 5 s to remove the culture medium.…”

Section: X-ray Microanalysismentioning

confidence: 99%

Biphasic behavior of changes in elemental composition during staurosporine-induced apoptosis

et al. 2005

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Although the identification of events that occur during apoptosis is a fundamental goal of apoptotic cell death research, little is know about the precise sequence of changes in total elemental composition during apoptosis. We evaluated total elemental composition (Na, Mg, P, Cl, S, and K) in relation to molecular and morphological features in human U937 cells induced to undergo apoptosis with staurosporine, an intrinsic pathway activator. To evaluate total elemental content we used electron probe X-ray microanalysis to measure simultaneously all elements from single, individual cells. We observed two phases in the changes in elemental composition (mainly Na, Cl and K). The early phase was characterized by a decrease in intracellular K (P<0.001) and Cl (P<0.001) content concomitant with cell shrinkage, and preceded the increase in proteolytic activity associated with the activation of caspase-3. The later phase started with caspase-3 activation, and was characterized by a decrease in the K/Na ratio (P<0.001) as a consequence of a significant decrease in K and increase in Na content. The inversion of intracellular K and Na content was related with the inhibition of Na+/K+ ATPase. This later phase was also characterized by a significant increase (P<0.001) in intracellular Cl with respect to the early phase. In addition, we found a decrease in S content and an increase in the P/S ratio. These distinctive changes coincided with chromatin condensation and DNA fragmentation. Together, these findings support the concept that changes in total elemental composition take place in two phases related with molecular and morphological features during staurosporine-induced apoptosis.

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“…Marshall and Condron showed that the depth from which 90% of the emitted x‐rays from all elements in a FH sample were detected was approximately 2 μm and that the lateral resolution was also about 2 μm. Measurements of electron penetration in FD cultured cells (Fernandez–Segura et al ., 1999) have yielded similar estimates of depth resolution to those calculated here for FD retina, however, the estimate of depth resolution (17 μm) in FD retina at 15 kV contrasts markedly with a depth resolution of 0.5 μm at 10 kV suggested by Liang et al . (2004) and Crewther et al .…”

Section: Resultsmentioning

confidence: 99%

Sequential quantitative X‐ray elemental imaging of frozen‐hydrated and freeze‐dried biological bulk samples in the SEM

Marshall

Goodyear

2011

Journal of Microscopy

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SummaryPlaned frozen-hydrated (FH) bulk biological samples of chicken retina were analysed by X-ray elemental imaging in a scanning electron microscope and reanalysed after freeze-drying in the microscope column. Sequential elemental imaging of the same bulk sample in this way provides improved information on element distributions. There was no evidence of element redistribution during the freeze-drying process. Quantitative elemental images were obtained and interpreted to deduce relative and absolute element concentrations in different regions of the retina. Water concentrations were determined from the difference in oxygen concentrations at 15 kV and 5 kV in FH and freeze-dried (FD) samples, respectively. Two accelerating voltages were used to maintain similar X-ray excitation volumes. Water concentrations were also estimated by relating measured oxygen concentration in FH samples to the concentration of oxygen in solutions of a generalized protein in water and by comparing concentrations of phosphorous or sulphur in the FH and FD states.

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A procedure to prepare cultured cells in suspension for electron probe X‐ray microanalysis: application to scanning and transmission electron microscopy

Cited by 11 publications

References 21 publications

Changes in intracellular sodium, chlorine, and potassium concentrations in staurosporine‐induced apoptosis

Changes in intracellular sodium, chlorine, and potassium concentrations in staurosporine‐induced apoptosis

Biphasic behavior of changes in elemental composition during staurosporine-induced apoptosis

Sequential quantitative X‐ray elemental imaging of frozen‐hydrated and freeze‐dried biological bulk samples in the SEM

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