Measurement of the cytosolic free calcium ion concentration of individual lymphocytes by microfluorometry using quin 2 or fura-2.

Komada, Hiroshi; Nakabayashi, Hiroshi; Nakano, Hiroshi; Hara, Masayuki; Yoshida, Toshimichi; Takanari, Hideki; Izutsu, Kosaku

doi:10.1247/csf.14.141

Cited by 9 publications

(6 citation statements)

References 11 publications

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“…Monocytes, U937 cells, monocyte-depleted (mixed) B and T cells, and murine CTLL cell line stably transfected with human Ib4R` (CTLL-hu-IL4-R) were loaded with the fluorescent intracellular Ca z § chelator dye, Fura-2, by incubating cells with 3 gM Fura-2 acetoxymethyl ester (Fura-2-AM) (Molecular Probes, Inc., Eugene, OR.) for 30 min at 37~ in Fura buffer as previously described (29)(30)(31). Fura buffer is composed of(in raM): 140 NaC1, 2.5 KC1, 2 CaC12, 5 MgC12, 5 glucose, and 10 Hepes, pH 7.2.…”

Section: Methodsmentioning

confidence: 99%

Interleukin 4 receptor signaling in human monocytes and U937 cells involves the activation of a phosphatidylcholine-specific phospholipase C: a comparison with chemotactic peptide, FMLP, phospholipase D, and sphingomyelinase.

Zhu

et al. 1994

The Journal of Experimental Medicine

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Interleukin 4 (IL-4) diminishes cytokine activation of human macrophage. IL-4 binding to monocyte IL-4R is associated with protein kinase C (PKC) translocation to a nuclear fraction. The cleavage of diacyglycerol (DAG), an activator of PKC, from membrane phospholipids was investigated to define the proximal events of IL-4R signaling. IL-4 induced a statistically significant time-and dose-dependent generation of DAG. The IL-4-triggered production of DAG was not derived from phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis, since neither cytosolic calcium flux nor liberation of inositol phosphates was detected in response to IL-4. Experiments were performed using [14C- methyl]choline-labeled U937 cells and monocytes to determine whether IL- 4R activated phospholipase C (PLC), PLD, or PLA2 to use membrane phosphatidylcholine (PC) to form DAG. IL-4 induced a time- and dose- dependent increase of phosphocholine (pchol) with concomitant degradation of membrane PC (p < 0.05 compared with control). The finding that the peak reduction of PC was equivalent to peak production of pchol suggested that IL-4R signaling involved the activation of a PC- specific PLC. Changes in choline (chol) or lyso-PC and glycerolphosphocholine, the respective products of PC cleavage by PLD or PLA2, were not detected in IL-4-treated cells. In contrast, exogenous PLD induced an increase in chol and concomitant loss of membrane PC. Additional investigation suggested that IL-4R signaling does not involve PLD. In cells labeled with L-lyso-3-PC 1-[1- 14C]palmitoyl, PLD but not IL-4, increased the production of phosphatidic acid (PA) and phosphatidyl-ethanol when pretreated with ethanol. Propranolol, an inhibitor of phosphatidate phosphohydrolase, and calyculin A, a phosphatase 1 and 2A inhibitor, blocked DAG production in response to FMLP but not to IL-4. In propranolol pretreated cells, PMA but not IL-4 triggered the production of PA and lowered the amount of DAG. Evidence that PLA2 is not coupled to IL-4R is the detection of arachidonate production in response to FMLP but not to IL-4. Furthermore, IL-4R is not coupled to sphingomyelinase (SMase) since IL-4, unlike exogenous SMase, did not generate ceramide but induced the hydrolysis of PC to pchol that was comparable to exogenous PLC. In summary, IL-4R signaling in monocytes and U937 cells involves PLC and not PLD, PLA2, or SMase, and it uses PC and not PIP2 to form DAG.

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Section: Methodsmentioning

confidence: 99%

Interleukin 4 receptor signaling in human monocytes and U937 cells involves the activation of a phosphatidylcholine-specific phospholipase C: a comparison with chemotactic peptide, FMLP, phospholipase D, and sphingomyelinase.

Zhu

et al. 1994

The Journal of Experimental Medicine

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“…Consequently, most measurements of intracellular calcium or Ca 2+ imaging are performed using fluorescence indicators (1)(2)(3), such as Quin-2 and Fura-2, which change intensity in response to Ca 2+ . The second-generation dye Fura-2 is currently preferred for a variety of chemical and biochemical reasons (3,4). The primary advantage of Fura-2 over Quin-2 appears to be the shift in its excitation wavelength in response to Ca 2+ (5-7), which allows calculation of the calcium concentration from the ratio of the fluorescence intensities at two excitation wavelengths, thus providing a measure of the [Ca 2+ ] which is independent of the probe concentration.…”

Section: Introductionmentioning

confidence: 99%

“…In order to circumvent these difficulties, a new series of calcium probes has been developed by Molecular Probes (11). These probes, called Calcium Green (CaG), 4 Calcium Orange (CaO), and Calcium Crimson (CaC), have absorption maximum at 508, 552, and 590 nm, respectively, and still longer excitation wavelengths could be used on the longwavelength sides of the absorption. The chemical structures of these probes have only recently become available (12) and are shown in Scheme I.…”

Section: Introductionmentioning

confidence: 99%

Calcium imaging using fluorescence lifetimes and long-wavelength probes

1992

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We describe imaging of calcium concentrations using the long-wavelength Ca(2+) indicators, Calcium Green, Orange, and Crimson. The lifetimes of these probes were measured using the frequency-domain method and were found to increase from 50% to severalfold in response to calcium. The two-dimensional images of the calcium concentration were obtained using a new apparatus for fluorescence lifetime imaging (FLIM). We also describe procedures to correct for the position-dependent frequency response of the gain-modulated image intensifier used in the FLIM apparatus. Importantly, the FLIM method does not require the probe to display shifts in the excitation or emission spectra. Using the FLIM method, calcium imaging is possible using probes which display changes in lifetime in response to calcium. Consequently, calcium imaging is possible with excitation wavelengths ranging from 488 to as long as 620 nm, where autofluorescence and/or photochemical damage is minimal. These probes are also suitable for calcium measurements of single cells using lifetime-based flow cytometry.

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“…Evidence has accumulated for the pivotal role of calcium ions (Ca 2+) in lymphocyte stimulation: 1) extracellular Ca 2÷ is essential for the mitogenesis [1,19] ; 2) mitogenic lectins induce early increase both in 45Ca2+ uptake [12,19] and in cytoplasmic free calcium ion concentration [13,19,20,37,38]; and 3) Ca2+-ionophore A23187 [23,24] and ionomycin [20] have a mitogenic potency, while calciumchannel blockers inhibit lectin-induced stimulation [4,5,26,27].…”

Section: Introductionmentioning

confidence: 99%

Lymphocyte calmodulin and its participation in the stimulation of T lymphocytes by mitogenic lectins

Nakabayashi

Komada

Yoshida

et al. 1992

Biology of the Cell

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Calmodulin was purified from human tonsillar lymphocytes utilizing calcium-dependent binding of calmodulin to fluphenazine-Sepharose. The molecular weight and phosphodiesterase activation of the lymphocyte calmodulin were very similar to those of purified bovine brain calmodulin. Trifluoperazine (TFP), a calmodulin inhibitor, suppressed lymphocyte stimulation as assessed by 3H-thymidine incorporation into DNA of lectin-stimulated lymphocytes. TFP had no effect on the early 45Ca2+ uptake induced by mitogenic lectins, although this latter was inhibited by verapamil which also suppressed the 3H-thymidine incorporation. The results are in keeping with the interpretation that the inhibition of T cell stimulation by TFP was not due to suppression of Ca2+ uptake, but due to inactivation of Ca(2+)-calmodulin complex which might be formed subsequent to Ca2+ entry into the cell.

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Measurement of the cytosolic free calcium ion concentration of individual lymphocytes by microfluorometry using quin 2 or fura-2.

Cited by 9 publications

References 11 publications

Interleukin 4 receptor signaling in human monocytes and U937 cells involves the activation of a phosphatidylcholine-specific phospholipase C: a comparison with chemotactic peptide, FMLP, phospholipase D, and sphingomyelinase.

Interleukin 4 receptor signaling in human monocytes and U937 cells involves the activation of a phosphatidylcholine-specific phospholipase C: a comparison with chemotactic peptide, FMLP, phospholipase D, and sphingomyelinase.

Calcium imaging using fluorescence lifetimes and long-wavelength probes

Lymphocyte calmodulin and its participation in the stimulation of T lymphocytes by mitogenic lectins

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