Fast intracellular pH determination in single cells by flow-cytometry

Valet, G.; Raffael, A.; Moröder, Luis; Wünsch, Erich; Ruhenstroth‐Bauer, G.

doi:10.1007/bf01047331

Cited by 91 publications

(46 citation statements)

References 7 publications

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“…Cells were loaded with the fluorescent pH sensitive dual emission dye dicyanohydroquinone (DCH) [13] by incubation with the esterified form diacetoxythalonitrile (ADB:Molecular Probes, Oregon, USA, or Paesel, FRG) for 5 min. The experiments were done in Balanced Salt Solutions containing 140 mM NaCI, 10 mM KCI, 2 mM CaCI2, 2 mM MgCI2, 10 mM glucose, 10 mM Hepes buffer at pH 7.4.…”

Section: Methodsmentioning

confidence: 99%

Growth stimulation by bombesin does not involve activation of Na⁺/H⁺ exchange in chick embryo otic vesicles in vitro

Gillespie¹,

Giráldez

Greenwell³

1989

FEBS Letters

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The effect of the mitogen bombesin on pHi in cells of the chick otic vesicle was studied using dual wavelength microspectrofluorimetric techniques. The results show that the bombesin did not produce an immediate alkalinization in quiescent otic vesicle cells nor a long-term change in pH~ in vesicles reactivated to grow in culture. The recovery of pH i from an acid load, caused by an NH 4 pulse, involved both Na÷/H ÷ exchange and Na÷:HCO3 influx mechanisms, neither of which was influenced by bombesin. These observations do not fit with a general model whereby pH~ is a universal signal for DNA replication and cell proliferation.

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Section: Methodsmentioning

confidence: 99%

Growth stimulation by bombesin does not involve activation of Na⁺/H⁺ exchange in chick embryo otic vesicles in vitro

Gillespie¹,

Giráldez

Greenwell³

1989

FEBS Letters

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“…Our pHi measurements as well as other data [8] demonstrate that the latter possibility takes place: at pHo 6.9 maintained in ascitic fluid, pHi proved to be equal to 6.6-6.7.…”

Section: Resultsmentioning

confidence: 48%

Acidification of the interior of Ehrlich ascites tumor cells by nigericin inhibits DNA synthesis

Margolis¹,

Rozovskaja²,

Skulachev³

1987

FEBS Letters

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In Ehrlich ascites carcinoma cells, acidification of the cytoplasm down to pH 6.2–6.3 arrests DNA synthesis. Such acidification can be obtained by decreasing the pH outside the cell or, alternatively, by addition of a micromolar concentration of the K+/H+ antiporter nigericin. Thus, nigericin may be regarded as a new type of cytostatic, the effect of which is mediated by alteration of the intracellular pH.

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“…Staining and analysis were as described for lichens. Samples were analyzed using the method described by Valet et a1 (24). Briefly, the intracellular pH was measured by flow cytometry using the ratio of emissions produced by 457-and 488-nm excitation.…”

Section: Staining Proceduresmentioning

confidence: 99%

“…Controls were freshly collected lichens and esterases break the ester bonds, producing fluorescein, which is retained in viable cells. The intensity of fluorescence depends on esterase activity in the cells (51, intracellular pH (24,25), and integrity of cell membranes (16,ZO were gated on chlorophyll fluorescence to exclude all non-chlorophyluntreated greenhouse specimens. Bold's basal medium (7) was used as a source of ions presumably present in natural precipitation.…”

mentioning

confidence: 99%

Flow cytometric measurement of pollutant stresses on algal cells

Berglund

Eversman

1988

Cytometry

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The lichen Usnea fuluoreugens (Ras.) Ras. was treated with four pH levels (5.5, 4.5, 3.5, and 2.5) of simulated acid rain (sulfuric acid, nitric acid, and a 1:l combination of both) and automobile exhaust. The samples were dissociated and analyzed by a Becton-Dickinson FACS 440 flow cytometer. Analyses included measurement of chlorophyll autofluorescence and fluorescence due to uptake of fluorescein diacetate (FDA) and calcofluor white M2R (CFW). Cell parameters measured were esterase activity (FDA), membrane permeability (FDA, CFW), and intracellular pH (FDA). Mean fluorescence intensity from FDA staining and numbers of events were incorporated with autofluorescence information to produce a "stress index" of relative cell stress. Results indicated that highly stressed samples (lower pH treatments and greater exposure to exhaust) exhibited a low "stress index'' of FDA fluorescence.Key terms: Flow cytometry, lichen, pollutants, fluorescein diacetate, calcofluor white M2RThe use of flow cytometry in plant sciences has been limited. Plant scientists have used flow cytometry in determining protoplast viability in tissue culture (11,19) for hybridization experiments (1,18) and for characterizing DNA content during the cell cycle (8,17). Dead, infected, and healthy protoplasts from cucumber plants infected with parasitic fungus showed differences in fluorescence properties when stained with fluorescein diacetate (Berglund, Strange, and Strobel, unpublished data). Lipid, DNA, and chlorophyll content have been examined in algae using flow cytometry (2,4,9,15,23,26,27).Lichens are symbiotic organisms composed of an alga and a fungus. Their sensitivity to pollutants has been extensively documented (10,12). Simulated acid precipitation has caused reduction of photosythesis rates (14,21,22) and reduction of nitrogen fixation in lichen species (6,221. Chlorophyll autofluorescence in lichens, another indicator of cellular conditions, has been shown to shift from red toward green due to pollutant stress (13). or permeablized cells more intensely than live cells (3).To our knowledge this is the first report of analyses of pollutant damage to plant or algal cells using flow cytometry. This flow cytometric analysis of autofluorescence characteristics and fluorescence produced by FDA staining indicates that lichen algal cells can be monitored for stress owing to acid precipitation and automobile exhaust. [Mirbell Franco) with the fruticose lichen Usnea fulvoreagens (Ras.) Ras. were collected in the Gallatin National Forest (Gallatin Co., Montana) and suspended in partial shade in a greenhouse. The temperature and sunlight available to the samples were dictated by normal weather patterns. The first experiment began in early summer and ended in late fall, so considerable climatic variations were experienced. The lichens and branches were saturated three times a week for 20 weeks with simulated acid precipitation. pH levels of sulfuric acid, Fluorescein diacetate (FDA) is a noduorescent corn-nitric acid, and a 1:lmixtm-e of both aci...

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Fast intracellular pH determination in single cells by flow-cytometry

Cited by 91 publications

References 7 publications

Growth stimulation by bombesin does not involve activation of Na⁺/H⁺ exchange in chick embryo otic vesicles in vitro

Growth stimulation by bombesin does not involve activation of Na⁺/H⁺ exchange in chick embryo otic vesicles in vitro

Acidification of the interior of Ehrlich ascites tumor cells by nigericin inhibits DNA synthesis

Flow cytometric measurement of pollutant stresses on algal cells

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Fast intracellular pH determination in single cells by flow-cytometry

Cited by 91 publications

References 7 publications

Growth stimulation by bombesin does not involve activation of Na+/H+ exchange in chick embryo otic vesicles in vitro

Growth stimulation by bombesin does not involve activation of Na+/H+ exchange in chick embryo otic vesicles in vitro

Acidification of the interior of Ehrlich ascites tumor cells by nigericin inhibits DNA synthesis

Flow cytometric measurement of pollutant stresses on algal cells

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Growth stimulation by bombesin does not involve activation of Na⁺/H⁺ exchange in chick embryo otic vesicles in vitro

Growth stimulation by bombesin does not involve activation of Na⁺/H⁺ exchange in chick embryo otic vesicles in vitro