A flow cytometric procedure for the isolation of antigenically responsive human basophils

Pendy, Luann; Arra, Susan J.; Anselmino, Lisa M.; Thomas, Larry L.

doi:10.1016/0022-1759(86)90102-x

Cited by 8 publications

(2 citation statements)

References 13 publications

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“…Several flow cytometric methods for the study and the quantification of basophil activation have been previously described (1,3,14,15,17). It has been reported that stimulation of basophils by activating agents could alter the expression of many basophil surface antigens ( 2 0 ) , including CD45 (2), CD63 (lo), and CD11 and Leu8 (2).…”

Section: Discussionmentioning

confidence: 99%

Flow cytometric monitoring of allergen induced basophil activation

et al. 1995

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Human basophils express many clustered differentiation antigens (CD), including CD45; however, none are speciac for these cells. In a previous study, we described a two-color immunofluorescence procedure, employing antibodies to CD45 and I@ for the cytometric evaluation of basophils. In Human basophils are quantitatively the least important population of circulating leukocytes. Nevertheless, they are specialized effector cells of the immune system and play an important role in host defense mechanisms and allergic reactions. The binding of allergens to specific IgE fixed on the basophil membrane via the FceRI receptor triggers cellular activation, degranulation, and the release of potent mediators, including histamine (4,6,7). Basophil activation is usually quantified by the measurement of secreted histamine (4,16) and by direct microscopic examination of the percentage of degranulated cells (6). This latter method is very simple but, due to the low frequency of circulating basophils, it is not of current use as it is poorly reliable.In a previous study (5) we showed that the CD45dimIgEb'igh' phenotype could be used for the flow cytometric evaluation of human basophils. However, when these cells were incubated in the presence of calcium and magnesium, three anti-IgE concentration-dependent modifications were observed: (i) a decrease in the number of detectable basophils, (ii) a decrease in IgE mean fluorescence intensity (MFI), and (iii) an increase in CD45 MFI. In the present work, the modulation of CD45 and IgE antigen expression induced on basophils by allergen and calcium ionophore is studied and a flow cytometric approach is described that can be used for the quantification of basophil activation. MATERIAL AND METHODSCell Preparation Peripheral blood from 10 nonatopic and 25 atopic adult volunteers was collected on EDTA. Atopic individuals were selected based on clinical allergic symptoms, positivity to at least one common aeroallergen (skin test), and the presence of specific IgE to this allergen. Seven patients were allergic to Dermutopbagoides pteronyssintcs (DP), 1 1 were allergic to Ductylis glomautu (DG), 4 were allergic to cat dander, and 3 were allergic to birch pollen. The blood, diluted 1/2 in phosphate buffer saline (PBS pH 7.2, BioMerieux, MarcyI'Etoile, France), was layered over a cushion of Percoll (Pharmacia, Uppsala, Sweden) adjusted to a specific gravity of 1.080 g/ml and centrifuged at 300 X g for 20 min at room temperature. The leukocyte-rich layer was recovered and washed in PBS, and the cells were finally resuspended in PBS supplemented with 0.5% bovine serum albumin

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Section: Discussionmentioning

confidence: 99%

Flow cytometric monitoring of allergen induced basophil activation

et al. 1995

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“…In previous flow cytometric studies, FITC-IgE anti-DNP (13) and Bsp-1 (3) MAbs were used for the isolation of basophils and their phenotypic analysis. To our knowledge these antibodies are unfortunately not available for general use.…”

Section: Discussionmentioning

confidence: 99%

Flow cytometric evaluation of human basophils

et al. 1993

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The expression of CD45 and IgE cell surface antigens on human leucocytes was studied by flow cytometry. More than 80% of sorted cells that expressed low CD45 (CD45dim) and high IgE (IgEbright) antigen site density were identified as basophils. Immunomagnetic depletion of the CD45d'"-IgEbT'ght cell subset by a biotin-coupled anti-IgE antibody and streptavidin-coated magnetic beads was greater than 90%, and more than 80% of cells binding significant numbers of beads exhibited the morphological characteristics of basophils. Interestingly, when the cell staining was performed in the presence of calcium and magnesium, we observed a significant increase of CD45 and an equivalent decrease of IgE cell surface expression, as well as an IgE concentration dependent diminution of the number of CD45d'"-IgEbnght cells.

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Eosinophil granule proteins in peripheral blood granulocytes

Abu-Ghazaleh

Dunnette

Loegering

et al. 1992

Journal of Leukocyte Biology

169

121

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Eosinophils contain four principal cationic proteins, major basic protein (MBP), eosinophil-derived neurotoxin (EDN), eosinophil cationic protein (ECP), and eosinophil peroxidase (EPO). To determine the quantities of these proteins in granulocytes and whether they are specific to eosinophils, their concentrations in lysates of human granulocytes were measured using specific radioimmunoassays. The effect of different methods for eosinophil lysis on the recovery of the proteins was also studied. Maximal recovery occurred at pH 2 for MBP and pH 5.6 for the other granule proteins. The proteins cosedimented with eosinophils and their concentrations (mean +/- SEM) in ng/10(6) eosinophils (and in nM/10(6) eosinophils) were: MBP, 8,982 +/- 611 (641.6); EDN, 3,283 +/- 116 (178.4); ECP, 5,269 +/- 283 (250.9); and EPO, 12,174 +/- 859 (171.5). Basophils from a normal person contained (in ng/10(6) cells) MBP, 2,374; EDN, 214; ECP, 77; and EPO, 17. Highly purified neutrophils contained (in ng/10(6) cells) MBP, 3 +/- 0.5; EDN, 72 +/- 9; and ECP, 50 +/- 12. Therefore we conclude that these proteins are mainly expressed in eosinophils, but that certain ones are present in basophils and neutrophils.

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A flow cytometric procedure for the isolation of antigenically responsive human basophils

Cited by 8 publications

References 13 publications

Flow cytometric monitoring of allergen induced basophil activation

Flow cytometric monitoring of allergen induced basophil activation

Flow cytometric evaluation of human basophils

Eosinophil granule proteins in peripheral blood granulocytes

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