Intracellular pH mapping with SNARF-1 and confocal microscopy. I: A quantitative technique for living tissue and isolated cells

Cody, Stephen H.; Dubbin, Philip N.; Beischer, Andrew D.; Duncan, Noel D.; Hill, John S.; Kaye, Andrew H.; Williams, David A.

doi:10.1016/0968-4328(93)90034-x

Cited by 38 publications

(19 citation statements)

References 18 publications

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“…By attaching such a linker to SNARF-1 and using it with an expressed HaloTag fusion construct, we are able to precisely target this pH sensor subcellularly and measure pH. We chose the SNARF-1 dye because it is a member of a family of pH sensors that continue to be standard in the field since they were first introduced in the early 1990s (11,12). These dyes are widely used for pH measurement because they show distinct emission bands based on protonation, they are conveniently excited at 488 or 515 nm with argon ion lasers, and they exhibit pKa values within physiological range.…”

Section: Introductionmentioning

confidence: 99%

Direct pH Measurements by using Subcellular Targeting of 5(and 6-) Carboxyseminaphthorhodafluor in Mammalian Cells

et al. 2009

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As a means of reliably measuring intracellular pH, we have precisely targeted 5(and 6-) carboxyseminaphthorhodafluor to cellular subcompartments. This was accomplished by combining the well-established pH-sensitive dye with a protein-based reporter system. When expressed in cells, the reporter protein is designed to covalently bind ligands composed of a functional group and a reactive linker. In order to make a pH-sensitive ligand, we chemically coupled the pH sensor to a reactive linker. Several ligands of differing linker lengths were made and tested for their pH responsiveness in vitro. The most responsive of these ligands was then evaluated for its efficacy in live cell labeling and its use as an intracellular pH sensor for ratiometric confocal microscopy. Here we show that we could target this pH sensor within mammalian cells exclusively to either the nucleus or cytoplasm. Exhibiting the versatility of this reporter technology, we were also able to specifically limit pH sensor labeling to within the trafficking pathway of integrins and directly measure pH of this environment. Results correspond well with previously published reports. Both the simplicity and flexibility of the technology used in this study make possible the development of diverse targeted microenvironmental sensors or other moieties of interest.

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Section: Introductionmentioning

confidence: 99%

Direct pH Measurements by using Subcellular Targeting of 5(and 6-) Carboxyseminaphthorhodafluor in Mammalian Cells

et al. 2009

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“…For these reasons, the tumour spheroid model was selected to investigate further the potential use of labelled MAbs in combination with hyperthermia, in particular as it might apply to the treatment of micrometastatic disease with 21'At-labelled MAbs. Although several investigators have achieved greater penetration of small molecules into spheroids by performing incubations under hypothermic conditions (Cody et al, 1993;Wartenberg and Acker, 1995), these results probably are not germane to our attempt to use hyperthermia to achieve the same goal. In these studies, increased penetration of fluorescent dyes such as SNARF-1 into spheroids was accomplished by performing incubations at 8-10°C.…”

Section: Discussionmentioning

confidence: 72%

Cytotoxicity of α-particle-emitting astatine-211-labelled antibody in tumour spheroids: no effect of hyperthermia

Hauck

Larsen²,

Welsh³

et al. 1998

Br J Cancer

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Summary The high linear energy transfer, a-particle-emitting radionuclide astatine-211 (21'At) is of interest for certain therapeutic applications; however, because of the 55-to 70-jim path length of its a-particles, achieving homogeneous tracer distribution is critical. Hyperthermia may enhance the therapeutic efficacy of a-particle endoradiotherapy if it can improve tracer distribution. In this study, we have investigated whether hyperthermia increased the cytotoxicity of an 21'At-labelled monoclonal antibody (MAb) in tumour spheroids with a radius (approximately 100 gm) greater than the range of 21'At a-particles. Hyperthermia for 1 h at 420C was used because this treatment itself resulted in no regrowth delay. Radiolabelled chimeric MAb 81C6 reactive with the extracellular matrix antigen tenascin was added to spheroids grown from the D-247 MG human glioma cell line at activity concentrations ranging from 0.125 to 250 kBq ml-'. A significant regrowth delay was observed at 125 and 250 kBq ml-' in both hyperthermia-treated and untreated spheroids. For groups receiving hyperthermia, no increase in cytotoxicity was seen compared with normothermic controls at any activity concentration. These results and those from autoradiographs indicate that hyperthermia at 420C for 1 h had no significant effect on the uptake or distribution of this antitenascin MAb in D-247 MG spheroids.Keywords: monoclonal antibody; hyperthermia; spheroid; astatine-21 1; a-emitterThe use of a-particle-emitting radionuclides for endoradiotherapy has several potential advantages compared with 1-emitters. The high linear energy transfer (LET) of a-particles makes them highly cytotoxic, rendering even hypoxic cells vulnerable. In addition, a-particles have relatively short effective path lengths in tissue, decreasing the radiation delivered to normal tissues located in close proximity to the tumour. For example, the mean path length of the a-particles emitted by 7.2-h half-life 21'At is 55-70 gm compared with 800 gm for the 5-particles emitted by '3'I (Gaze et al, 1992;. In contrast, the short a-particle range is potentially problematic because of the necessity for achieving homogeneous distribution of the radionuclide in the tumour for effective therapy. The limited range of a-particles has led investigators to focus therapeutic applications in which the tumour is present in thin sheets, such as neoplastic meningitis and peritoneal metastasis. The efficacy of 21'At-labelled monoclonal antibodies (MAbs) in a rat model of neoplastic meningitis has been encouraging .Micrometastatic disease could offer another potential application of a-emitters if sufficiently homogeneous tracer distribution could be achieved. The determination of the most appropriate radionuclide for the treatment of small volume disease has been the focus of both experimental and theoretical studies (Humm and Cobb, 1990;Langmuir et al, 1990; 1992a higher absorbed dose fraction within the tumour compared with long-range 3-emitters such as 90Y, particularly for lesions of less than...

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“…Fluo-3/AM can diffuse across plasma membranes of pollen at high (37°C) or low temperatures (4°C). There have been previous explanations that high or low temperatures could reduce activity of extracellular esterases and minimize extracellular hydrolysis of esters of Fluo-3 (Cody et al 1993;Zhang et al 1998).…”

Section: Discussionmentioning

confidence: 99%

Fast loading ester fluorescent Ca2+ and pH indicators into pollen of Pyrus pyrifolia

Jiang

Z³

et al. 2011

J Plant Res

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Loading of Ca(2+)-sensitive fluorescent probes into plant cells is an essential step to measure activities of free Ca(2+) ions in cytoplasm with a fluorescent imaging technique. Fluo-3 is one of the most suitable Ca(2+) indicators for CLSM. We loaded pollen with fluo-3/AM at three different temperatures. Fluo-3/AM was successfully loaded into pollen at both low (4°C) and high (37°C) temperatures. However, high loading temperature was best suited for pollen, because germination rate of pollen and growth of pollen tubes were relatively little impaired and loading time was shortened. Moreover, Ca(2+) distribution increased in the three apertures of pollen after hydration and showed a Ca(2+) gradient, similar to the tip of growing pollen tubes. The same protocol can be used with the AM-forms of other fluorescent dyes for effective labeling. When loading BCECF-AM into pollen at high temperature, the pollen did not show a pH gradient after hydration. Ca(2+) activities and fluxes had the same periodicity as pollen germination, but pH did not show the same phase and mostly lagged behind. However, the clear zone was alkaline when pollen tube growth was slowed or stopped and turned acidic when growth recovered. It is likely that apical pH(i) regulated pollen tube growth.

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Intracellular pH mapping with SNARF-1 and confocal microscopy. I: A quantitative technique for living tissue and isolated cells

Cited by 38 publications

References 18 publications

Direct pH Measurements by using Subcellular Targeting of 5(and 6-) Carboxyseminaphthorhodafluor in Mammalian Cells

Direct pH Measurements by using Subcellular Targeting of 5(and 6-) Carboxyseminaphthorhodafluor in Mammalian Cells

Cytotoxicity of α-particle-emitting astatine-211-labelled antibody in tumour spheroids: no effect of hyperthermia

Fast loading ester fluorescent Ca2+ and pH indicators into pollen of Pyrus pyrifolia

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