Optimization of a microplate reader method for the analysis of changes in mitochondrial membrane potential in Chlamydomonas reinhardtii cells using the fluorochrome JC-1

Harshkova, Darya; Zielińska, Elżbieta; Aksmann, Anna

doi:10.1007/s10811-019-01860-3

Cited by 10 publications

(4 citation statements)

References 29 publications

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“…The MMP measurement via JC-1 was carried out in the 96-multiwell black plates, following Harshkova et al [ 36 ] with a slight modification. Similar to the procedure described for labelling the mt-ROS, after 24 h of treatment with the methanolic extracts or isolated compounds, blank control, and positive control of metformin, the mitochondria of the adipocytes were stained with 3 µM of JC-1 for 30 min in a humidified incubator.…”

Section: Methodsmentioning

confidence: 99%

Antihyperglycemic Properties of Extracts and Isolated Compounds from Australian Acacia saligna on 3T3-L1 Adipocytes

et al. 2023

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Our early work indicated that methanolic extracts from the flowers, leaves, bark, and isolated compounds of Acacia saligna exhibited significant antioxidant activities in vitro. The overproduction of reactive oxygen species (ROS) in the mitochondria (mt-ROS) interfered with glucose uptake, metabolism, and its AMPK-dependent pathway, contributing to hyperglycemia and diabetes. This study aimed to screen the ability of these extracts and isolated compounds to attenuate the production of ROS and maintain mitochondrial function via the restoration of mitochondrial membrane potential (MMP) in 3T3-L1 adipocytes. Downstream effects were investigated via an immunoblot analysis of the AMPK signalling pathway and glucose uptake assays. All methanolic extracts effectively reduced cellular ROS and mt-ROS levels, restored the MMP, activated AMPK-α, and enhanced cellular glucose uptake. At 10 µM, (−)-epicatechin-6 (from methanolic leaf and bark extracts) markedly reduced ROS and mt-ROS levels by almost 30% and 50%, respectively, with an MMP potential ratio 2.2-fold higher compared to the vehicle control. (−)-Epicatechin 6 increased the phosphorylation of AMPK-α by 43%, with an 88% higher glucose uptake than the control. Other isolated compounds include naringenin 1, naringenin-7-O-α-L-arabinopyranoside 2, isosalipurposide 3, D-(+)-pinitol 5a, and (−)-pinitol 5b, which also performed relatively well across all assays. Australian A. saligna active extracts and compounds can reduce ROS oxidative stress, improve mitochondrial function, and enhance glucose uptake through AMPK-α activation in adipocytes, supporting its potential antidiabetic application.

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Section: Methodsmentioning

confidence: 99%

Antihyperglycemic Properties of Extracts and Isolated Compounds from Australian Acacia saligna on 3T3-L1 Adipocytes

et al. 2023

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“…In healthy cells, JC-1 dye could enter and aggregate in negatively charged mitochondria, spontaneously forming red fluorescent aggregates. In unhealthy or apoptotic cells, due to the loss of mitochondrial membrane potential, JC-1 dye entered into the mitochondria less, and the formation of aggregates was insufficient, thus maintaining its original green fluorescence [ 31 , 32 ]. Therefore, the red/green fluorescence ratio in mitochondria was an assessment of the state of the mitochondria.…”

Section: Resultsmentioning

confidence: 99%

Interrogation on the Cellular Nano-Interface and Biosafety of Repeated Nano-Electroporation by Nanostraw System

et al. 2022

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Cell perforation is a critical step for intracellular drug delivery and real-time biosensing of intracellular signals. In recent years, the nanostraws system has been developed to achieve intracellular drug delivery with minimal invasiveness to the cells. Repeated cell perforation via nano-system could allow delivery of multiple drugs into cells for cell editing, but the biosafety is rarely explored. In this work, a nanostraw-mediated nano-electroporation system was developed, which allowed repeated perforation of the same set of cells in a minimally invasive manner, while the biosafety aspect of this system was investigated. Highly controllable fabrication of Al2O3 nanostraw arrays based on a porous polyethylene terephthalate (PET) membrane was integrated with a microfluidic device to construct the nanostraw-electroporation system. The pulse conditions and intervals of nano-electroporation were systematically optimized to achieve efficient cells perforation and maintain the viability of the cells. The cells proliferation, the early apoptosis activities after nanostraw-electroporation and the changes of gene functions and gene pathways of cells after repeated nano-electroporation were comprehensively analyzed. These results revealed that the repeated nanostraw-electroporation did not induce obvious negative effects on the cells. This work demonstrates the feasibility of repeated nano-electroporation on cells and provides a promising strategy for future biomedical applications.

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“…JC‐1 has been widely used as a mitochondrial membrane potential indicator, and possess advantageous features as comparison to other cationic dyes. The selectively entering of JC‐1 into mitochondria leads to colour changes from red to green as the mitochondrial potential reduces [26]. In healthy cells with high mitochondrial potential (ΔΨ m ), JC‐1 instantly forms complexes recognised as J‐aggregates with high red fluorescence (590 nm) while in unhealthy cells the monomeric form JC‐1 emits green fluorescence at 530 nm owing to the low ΔΨ m .…”

Section: Methodsmentioning

confidence: 99%

Unveiling the cytotoxicity of phytosynthesised silver nanoparticles using Tinospora cordifolia leaves against human lung adenocarcinoma A549 cell line

Mittal

Pal

Sharma

et al. 2020

IET nanobiotechnol.

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Biosynthesis of silver nanoparticles (AgNPs) using plant extract is a cheap, easily accessible and natural process in which the phyto-constituents of the plants act as capping, stabilising and reducing agent. The present study explored the biosynthesis of AgNPs using aqueous leaf extract of Tinospora cordifolia and characterised via various techniques such as Fourier transform infrared, scanning electron microscopy, transmission electron microscopy (TEM), energy dispersive X-ray analysis and X-ray diffraction. Here, TEM confirmed the spherical morphology with 25-50 nm size of synthesised AgNPs. Further, anticancer efficiency of AgNPs synthesised using T. cordifolia leaves were evaluated against human lung adenocarcinoma cell line A549 by MTT, trypan blue assay, apoptotic morphological changes using Annexin V-FITC and Propidium iodide (PI), nuclear morphological changes by DAPI (4, 6-diamidino-2-phenylindole dihydrochloride) staining, reactive oxygen species generation and mitochondrial membrane potential determination. Results confirmed the AgNPs synthesised using T. cordifolia leaves are found to be highly toxic against human lung adenocarcinoma cell line A549.

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Optimization of a microplate reader method for the analysis of changes in mitochondrial membrane potential in Chlamydomonas reinhardtii cells using the fluorochrome JC-1

Cited by 10 publications

References 29 publications

Antihyperglycemic Properties of Extracts and Isolated Compounds from Australian Acacia saligna on 3T3-L1 Adipocytes

Antihyperglycemic Properties of Extracts and Isolated Compounds from Australian Acacia saligna on 3T3-L1 Adipocytes

Interrogation on the Cellular Nano-Interface and Biosafety of Repeated Nano-Electroporation by Nanostraw System

Unveiling the cytotoxicity of phytosynthesised silver nanoparticles using Tinospora cordifolia leaves against human lung adenocarcinoma A549 cell line

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