Cytofluorometric quantitation of 5-hydroxytryptamine in mast cells: an improved technique for the formaldehyde condensation reaction.

Enerbäck, Lennart; Gustafsson, Bertil; Mellblom, Lennart

doi:10.1177/25.1.556750

Cited by 29 publications

(8 citation statements)

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“…Previous studies have shown that the fluores cent formaldehyde reaction (13) can be used for specific and precise quantifica tion of 5-HT both in mast cells and in individual mast cell granules (12,19). To facilitate quantitation, we used mast cells with increased fluorescence intensity due to induced accumulation of 5-HT (12,13,19).…”

Section: Discussionmentioning

confidence: 99%

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A Cytofluorometric Analysis of Polymer-Induced Mast Cell Secretion

Gustafsson¹,

Enerbäck²

1980

Pathobiology

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Quantitative cytofluorometry was used to study the mechanism of mast cell secretion induced by polymyxin B in vitro. We measured 5-hydroxytryptamine (5-HT) and heparin in mast cells and individual mast cell granules, extruded from the cells or obtained from the interior of the cells by micromanipulation. Mast cell granules of untreated control cells contained both 5-HT and heparin. After polymyxin stimulation both intracellular and extruded mast cell granules contained heparin, but some intracellular and all extruded granules lacked 5-HT. These results indicate that the mast cell amines may be released from granules while they are still within the cellular domain. The mast cell granules did not release heparin whether extruded from the cell or not. The release of heparin from mast cells was thus determined by the number of extruded granules. Our results further suggest that the granules first released are those situated in the periphery of the cell and contain the largest quantities of amines and heparin. In agreement with the granule measurements, fluorescence measurements on stimulated mast cells showed a dose-dependent decrease of both heparin and 5-HT, but the 5-HT content decreased proportionally more than that of heparin.

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Section: Discussionmentioning

confidence: 99%

“…Quantitation o f 5-HT and Heparin 5-HT was quantitated after formaldehyde treatment according to Falck et al (15) with a modified and standardized technique as described previously (13). Heparin was quantitated with berberine binding after fixation in ethanol-acetic acid as described pre viously (11).…”

Section: Preparation O F Mast Cell Granulesmentioning

confidence: 99%

A Cytofluorometric Analysis of Polymer-Induced Mast Cell Secretion

Gustafsson¹,

Enerbäck²

1980

Pathobiology

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“…fluorescence, according to Falck et al (1962), and as described pre viously [Enerback et al, 1977b). Heparin and 5-HT release was cal culated as the percentage decrease in fluorescence intensity of mast cells incubated with polymyxin B in relation to control cells incu bated without polymyxin B.…”

Section: Microscopic Fluorometrymentioning

confidence: 99%

Fluorescent Berberine Binding as a Marker of Secretory Activity in Mast Cells

Berlin

Enerbäck

1983

Int Arch Allergy Immunol

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We have previously shown that the weakly fluorescent cationic dye, berberine, forms a strongly fluorescent complex with the heparin of fixed mast cells and can be used for cytofluorometric measurement of heparin in both mast cells and individual mast cell granules. Now, we report on the use of berberine as a vital stain, demonstrating the secretory activity of mast cells. At a dye concentration of 0.025%, and after membrane stabilization with polyethylene glycol, normal mast cells exclude the dye while mast cells stimulated to secretion with polymyxin B show a strongly fluorescent dye binding to individual cytoplasmic granules. The mean fluorescence intensity (reflecting the number of stained granules) of the cell populations increased with increasing polymyxin B concentrations up to 2.0 μg/ml, thereafter remaining constant up to 10 μg/ml. Fluorescence intensity after vital staining with berberine was compared both to release of heparin measured by berberine binding to fixed cells (reflecting exocytosis of mast cell granules) and also to histamine release. The results strongly suggest that berberine specifically stains secreting granules that are located within the domains of the cells but may have released histamine. Vital berberine staining combined with histamine assays and cytofluorometric measurements of heparin is therefore of great potential interest for analyzing the dynamics of mast cell secretion.

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“…5-HT was quantitated with the fluorescentformaldehyde technique [12,15]. Heparin was quantitated by berberine binding [10].…”

Section: Quantitation Of 5-ht and Heparinmentioning

confidence: 99%

“…By combining this method with the standard ized fluorescent formaldehyde reaction for 5-hydroxytryptamine (5-HT) [12] the con tent of both an amine and a suggested ma trix component in mast cell granules can be quantitated cytofluorometrically under dif ferent experimental conditions.…”

Section: Introductionmentioning

confidence: 99%

Cytofluorometric Analysis of Anaphylactic Secretion of 5-Hydroxytryptamine and Heparin from Rat Mast Cells

Gustafsson¹

1980

Int Arch Allergy Immunol

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Actively sensitized rat mast cells were stimulated to secretion in vitro by means of an anaphylactic IgE antigen reaction or with polymyxin B. Release of 5-hydroxytryptamine (5-HT) and heparin from the cells and from individual granules, extruded or displaced from stimulated cells with micromanipulation, was quantitated cytofluorometrically. Several similarities in action between the two secretagogues were found. All extruded and some intracellular granules from stimulated cells lacked 5-HT, and no heparin was released from the granules. Heparin secretion from mast cells was due to granule extrusion alone. Further, the proportions between 5-HT and heparin released from mast cells indicated that amine release, too, is mainly, but not exclusively, associated with overt granule exocytosis. The results suggested that the first extruded granules are the richest in both 5-HT and heparin. Antigen-induced secretion was not as vigorous as that induced by polymyxin and, unlike the latter, followed after a short lag of about 20 sec.

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Cytofluorometric quantitation of 5-hydroxytryptamine in mast cells: an improved technique for the formaldehyde condensation reaction.

Cited by 29 publications

References 0 publications

A Cytofluorometric Analysis of Polymer-Induced Mast Cell Secretion

A Cytofluorometric Analysis of Polymer-Induced Mast Cell Secretion

Fluorescent Berberine Binding as a Marker of Secretory Activity in Mast Cells

Cytofluorometric Analysis of Anaphylactic Secretion of 5-Hydroxytryptamine and Heparin from Rat Mast Cells

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