Synthesis and photophysical properties of new fluorinated benzo[c]xanthene dyes as intracellular pH indicators

Liu, Jixiang; Diwu, Zhenjun; Leung, Wai‐Yee

doi:10.1016/s0960-894x(01)00595-9

Cited by 120 publications

(82 citation statements)

References 25 publications

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“…In order to record the pH-dependent emission spectra, the cell extract comprising the hydrolyzed probe was diluted in buffers of various pH values. Fluorescence emission was recorded at an excitation wavelength of 543 nm as previously described (14). Spectral analyses clearly showed the presence of two pH-sen- (Fig.…”

Section: Resultsmentioning

confidence: 99%

Intracellular pH Distribution in Saccharomyces cerevisiae Cell Populations, Analyzed by Flow Cytometry

Valli

Sauer

Branduardi

et al. 2005

Appl Environ Microbiol

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Intracellular pH has an important role in the maintenance of the normal functions of yeast cells. The ability of the cell to maintain this pH homeostasis also in response to environmental changes has gained more and more interest in both basic and applied research. In this study we describe a protocol which allows the rapid determination of the intracellular pH of Saccharomyces cerevisiae cells. The method is based on flow cytometry and employs the pH-dependent fluorescent probe carboxy SNARF-4F. The protocol attempts to minimize the perturbation of the system under study, thus leading to accurate information about the physiological state of the single cell. Moreover, statistical analysis performed on major factors that may influence the final determination supported the validity of the optimized protocol. The protocol was used to investigate the effect of external pH on S. cerevisiae cells incubated in buffer. The results obtained showed that stationary cells are better able than exponentially grown cells to maintain their intracellular pH homeostasis independently of external pH changes. Furthermore, analysis of the intracellular pH distribution within the cell populations highlighted the presence of subpopulations characterized by different intracellular pH values. Notably, a different behavior was observed for exponentially grown and stationary cells in terms of the appearance and development of these subpopulations as a response to a changing external pH.

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Section: Resultsmentioning

confidence: 99%

Intracellular pH Distribution in Saccharomyces cerevisiae Cell Populations, Analyzed by Flow Cytometry

Valli

Sauer

Branduardi

et al. 2005

Appl Environ Microbiol

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“…pH i measurements. As described in detail previously (19,20), pH i in cultured islets was monitored by confocal microscopy using SNARF5 (carboxyseminaphthorhodofluor-5F-AM), a pH-sensitive fluorescent indicator with single excitation and dual emission (28). Before imaging, cultured islets were maintained in RPMI-1640 medium containing 5 mmol/l glucose for at least 48 h. Loading was done in basal KRBH, and islet pH i was measured in KRBH containing basal (2.8 mmol/l) or high (16.7 mmol/l) glucose.…”

Section: Methodsmentioning

confidence: 99%

Mechanisms of Time-Dependent Potentiation of Insulin Release

et al. 2006

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Time-dependent potentiation (TDP) of insulin release is normally absent in mice. However, we recently demonstrated that TDP occurs in mouse islets under conditions of forced decrease of intracellular pH (pH i ) associated with elevated NADPH؉H ؉ (NADPH) levels. Hence, TDP in mouse islets may be kept suppressed by neuronal nitric oxide (NO) synthase (nNOS), an NADPH-utilizing enzyme with alkaline pH optimum. To determine the role of nNOS in the suppression of TDP in mouse islets, glucose-induced TDP was monitored in mouse islets in which nNOS activity had been genetically removed or chemically inhibited and compared with the TDP response in wild-type mouse islets with and without forced intracellular acidification. Genetic deletion of nNOS was provided by an nNOS knockout (NOS-KO) mouse model, B6 -129S4-Nos1tm1Plh /J. To explore how nNOS inhibits TDP, we compared pH i and NADPH levels in wild-type and NOS-KO islets and monitored TDP with various components of the nNOS reaction added. Glucose normally does not produce TDP in wildtype mouse islets except under forced intracellular acidification. Remarkably, glucose produced strong TDP in NOS-KO islets and in wild-type islets treated with nNOS inhibitors. TDP in NOS-KO islets was not inhibited by the addition of NO, and NOS-KO islets exhibited a lower pH i than wild-type islets. The addition of arginine to wild-type islets also enabled glucose to induce TDP. Our results show that nNOS activity contributes to the absence of TDP in mouse islets putatively through depletion of intracellular arginine.

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“…Cytosolic pH (pH i ) was measured using the fluorescent pH-sensitive indicator, SNARF-5F (Liu et al, 2001). Cells in 96-well plates were incubated with SNARF-5F/AM (2 mM, 30 min), washed with HBS, and fluorescence (excitation at 561 nm, and emission at 580 and 640 nm) was measured using a FlexStation fluorescence plate reader.…”

Section: Measurement Of Cytosolic Phmentioning

confidence: 99%

Lysosomes shape Ins(1,4,5)P3-evoked Ca2+ signals by selectively sequestering Ca2+ released from the endoplasmic reticulum

López-Sanjurjo

Tovey

Prole

et al. 2013

Journal of Cell Science

123

111

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Summary Most intracellular Ca2+ signals result from opening of Ca 2+ channels in the plasma membrane or endoplasmic reticulum (ER), and they are reversed by active transport across these membranes or by shuttling Ca 2+ into mitochondria. Ca 2+ channels in lysosomes contribute to endo-lysosomal trafficking and Ca 2+ signalling, but the role of lysosomal Ca 2+ uptake in Ca 2+ signalling is unexplored. Inhibition of lysosomal Ca 2+ uptake by dissipating the H + gradient (using bafilomycin A 1 ), perforating lysosomal membranes (using glycyl-Lphenylalanine 2-naphthylamide) or lysosome fusion (using vacuolin) increased the Ca 2+ signals evoked by receptors that stimulate inositol 1,4,5-trisphosphate [Ins(1,4,5)P 3 ] formation. Bafilomycin A 1 amplified the Ca 2+ signals evoked by photolysis of caged Ins(1,4,5)P 3 or by inhibition of ER Ca 2+ pumps, and it slowed recovery from them. Ca 2+ signals evoked by store-operated Ca 2+ entry were unaffected by bafilomycin A 1 . Video-imaging with total internal reflection fluorescence microscopy revealed that lysosomes were motile and remained intimately associated with the ER. Close association of lysosomes with the ER allows them selectively to accumulate Ca 2+ released by Ins(1,4,5)P 3 receptors.

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Synthesis and photophysical properties of new fluorinated benzo[c]xanthene dyes as intracellular pH indicators

Cited by 120 publications

References 25 publications

Intracellular pH Distribution in Saccharomyces cerevisiae Cell Populations, Analyzed by Flow Cytometry

Intracellular pH Distribution in Saccharomyces cerevisiae Cell Populations, Analyzed by Flow Cytometry

Mechanisms of Time-Dependent Potentiation of Insulin Release

Lysosomes shape Ins(1,4,5)P3-evoked Ca2+ signals by selectively sequestering Ca2+ released from the endoplasmic reticulum

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