Characterization of Transport Activity of SLC11 Transporters in Xenopus laevis Oocytes by Fluorophore Quenching

Cinquetti, Raffaella; Imperiali, Francesca Guia; Bozzaro, Salvatore; Zanella, Daniele; Vacca, Francesca; Roseti, Cristina; Peracino, Barbara; Castagna, Michela; Bossi, Elena

doi:10.1177/24725552211004123

Cited by 4 publications

(3 citation statements)

References 44 publications

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“…Altered DMT1 expression leads to iron deposition in neuronal cells and neurodegenerative disorder [ 61 , 62 ]. In the present study, we identified DMT1 expression in mouse oocytes for the first time, which has been previously validated only in Xenopus oocytes [ 63 , 64 ]. Noteworthily, the protein level of DMT1 was elevated in aging ovaries and oocytes, such elevation may contribute to, at least partially, the redundancy of cytosolic Fe 2+ in oocytes.…”

Section: Discussionmentioning

confidence: 85%

Iron accumulation in ovarian microenvironment damages the local redox balance and oocyte quality in aging mice

Chen,

Zhang,

Tian

et al. 2024

Redox Biology

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Section: Discussionmentioning

confidence: 85%

Iron accumulation in ovarian microenvironment damages the local redox balance and oocyte quality in aging mice

Chen,

Zhang,

Tian

et al. 2024

Redox Biology

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“…The second problem is related to the autofluorescence of the oocytes, which changes from batch to batch, and, consequently, needs collecting for each experiment, as well as several reference samples. To solve the first problem, Cinquetti et al [ 135 ] developed a method in which the quenching of the fluorophore was used to indirectly monitor the translocation of substrates or ions, using a plate reader to collect a satisfactory number of data at the same time. The technique reported in [ 135 ] can be applied to measure the difference of the fluorescence in a cytoplasmic extract from the oocytes exposed or not to the substrate and is able to quench the fluorophore, indirectly measuring the uptake.…”

Section: Alternative Methods To Quantify the Functionalitymentioning

confidence: 99%

“…To solve the first problem, Cinquetti et al [ 135 ] developed a method in which the quenching of the fluorophore was used to indirectly monitor the translocation of substrates or ions, using a plate reader to collect a satisfactory number of data at the same time. The technique reported in [ 135 ] can be applied to measure the difference of the fluorescence in a cytoplasmic extract from the oocytes exposed or not to the substrate and is able to quench the fluorophore, indirectly measuring the uptake. In the same paper, autofluorescence was also investigated in this kind of assay.…”

Section: Alternative Methods To Quantify the Functionalitymentioning

confidence: 99%

The “www” of Xenopus laevis Oocytes: The Why, When, What of Xenopus laevis Oocytes in Membrane Transporters Research

et al. 2022

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After 50 years, the heterologous expression of proteins in Xenopus laevis oocytes is still essential in many research fields. New approaches and revised protocols, but also classical methods, such as the two-electrode voltage clamp, are applied in studying membrane transporters. New and old methods for investigating the activity and the expression of Solute Carriers (SLC) are reviewed, and the kinds of experiment that are still useful to perform with this kind of cell are reported. Xenopus laevis oocytes at the full-grown stage have a highly efficient biosynthetic apparatus that correctly targets functional proteins at the defined compartment. This small protein factory can produce, fold, and localize almost any kind of wild-type or recombinant protein; some tricks are required to obtain high expression and to verify the functionality. The methodologies examined here are mainly related to research in the field of membrane transporters. This work is certainly not exhaustive; it has been carried out to be helpful to researchers who want to quickly find suggestions and detailed indications when investigating the functionality and expression of the different members of the solute carrier families.

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A Microfluidic Chip on a Robotic Manipulator for Loading and Reloading of Oocytes

Liang,

Amaya,

Sugiura

et al. 2024

Advanced Intelligent Systems

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Loading individual oocytes is a critical step for injecting RNA, expressing heterologous proteins, performing electrophysiological measurements, and so on. However, existing methods remain a challenge for automatically loading multiple single oocytes into different locations. Herein, a novel microfluidic chip on a robotic manipulator (chip‐on‐robot) with feedback control for flexible manipulation of multiple oocytes within a large spatial range is proposed. The manipulator automatically controls the microfluidic chip to reach different locations based on imaging feedback. The microfluidic chip then utilizes the hydrodynamic focusing effect of the main channel to separate oocytes for individual loading or reloading under capacitive sensor feedback. The separation distance reaches approximately 16 times the oocyte diameter. Moreover, capacitive signal feedback on the number of oocytes for flow direction control ensures the separation of all oocytes. For close‐loop control of the loading/reloading process, image‐based oocyte detection is combined using deep learning to calculate the target position of the oocyte. Finally, an automatic sequence is achieved to load multiple single oocytes into a well chip by using the chip‐on‐robot. As a demonstration, the oocytes are reloaded into a specified location based on the conditions. The proposed chip‐on‐robot with feedback control has significant advantages in the micromanipulation of oocytes.

show abstract

Characterization of Transport Activity of SLC11 Transporters in Xenopus laevis Oocytes by Fluorophore Quenching

Cited by 4 publications

References 44 publications

Iron accumulation in ovarian microenvironment damages the local redox balance and oocyte quality in aging mice

Iron accumulation in ovarian microenvironment damages the local redox balance and oocyte quality in aging mice

The “www” of Xenopus laevis Oocytes: The Why, When, What of Xenopus laevis Oocytes in Membrane Transporters Research

A Microfluidic Chip on a Robotic Manipulator for Loading and Reloading of Oocytes

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