The physiological importance of mitochondrial calcium uptake, observed in processes such as ATP production, intracellular calcium signaling, and apoptosis, makes desirable a simple, straightforward way of investigating this event with unambiguous results. The following protocol uses a calcium-sensitive, membrane-impermeable fluorophore to monitor extra-mitochondrial calcium levels in the presence of permeabilized mammalian cells harboring activated mitochondria.
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http://dx.doi.org/10.21769/BioProtoc.2934 | DOI Listing |
Anal Chem
April 2021
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, P. R. China.
Aminopeptidase N (APN) is capable of cleaving N-terminal amino acids from peptides with alanine in the N-terminal position and plays a key role in the growth, migration, and metastasis of cancer. However, reliable information is hard to be obtained with the current APN-responsive molecular probes because the released fluorophores are cytoplasmic soluble and thus rapidly depart from the enzymatic reaction sites and spread out all over the cytoplasm. Here, we report a precipitated fluorophore, , which is completely insoluble in water and shows strong yellow solid emission when excited with a 405 nm laser.
View Article and Find Full Text PDFAnal Chem
February 2021
Molecular Science and Biomedicine Laboratory (MBL), State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Life Sciences, Hunan University, Changsha 410082, China.
Mitochondria-targeted fluorescent probes are highly important to obtain mitochondrial function information. However, the accuracy of the current mitochondria-targeted fluorescent probes is unsatisfactory owing to the following two reasons. In the first case, some probes that always have a mitochondria-targeting group, thus, would react with the analytes outside of mitochondria and enter mitochondria with the generated fluorophore signal, which leads to a false-positive result.
View Article and Find Full Text PDFJ Am Chem Soc
May 2020
AMED-CREST, Japan Agency for Medical Research and Development, 1-7-1 Otemachi, Chiyoda-ku, Tokyo 100-0004, Japan.
Single-molecule localization microscopy (SMLM) allows the reconstruction of super-resolution images but generally requires prior intense laser irradiation and in some cases additives to induce blinking of conventional fluorophores. We previously introduced a spontaneously blinking rhodamine fluorophore based on an intramolecular spirocyclization reaction for live-cell SMLM under physiological conditions. Here, we report a novel principle of spontaneous blinking in living cells, which utilizes reversible ground-state nucleophilic attack of intracellular glutathione (GSH) upon a xanthene fluorophore.
View Article and Find Full Text PDFBio Protoc
July 2018
Department of Biochemistry, Brandeis University, Waltham, USA.
The physiological importance of mitochondrial calcium uptake, observed in processes such as ATP production, intracellular calcium signaling, and apoptosis, makes desirable a simple, straightforward way of investigating this event with unambiguous results. The following protocol uses a calcium-sensitive, membrane-impermeable fluorophore to monitor extra-mitochondrial calcium levels in the presence of permeabilized mammalian cells harboring activated mitochondria.
View Article and Find Full Text PDFAdv Mater
July 2018
Institute of Rocket Force Medicine, State Key Laboratory of Trauma, Burns and Combined Injury, Third Military Medical University, Chongqing, 400038, China.
The endoplasmic reticulum (ER) stress signaling or unfolded protein response (UPR) is a common feature of many human diseases, including cancer. Excessive activation of ER stress directly induces cell death, holding a new promising strategy for the therapeutic intervention of cancer. Current ER-stress-inducing agents mainly target UPR components or proteasomes, which exert limited treatment efficacy and undesired side effects due to unselective ER stress and poor tumor-specific distribution.
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