Dataset Information

Agarose Spot as a Comparative Method for in situ Analysis of Simultaneous Chemotactic Responses to Multiple Chemokines.

ABSTRACT: We describe a novel protocol to quantitatively and simultaneously compare the chemotactic responses of cells towards different chemokines. In this protocol, droplets of agarose gel containing different chemokines are applied onto the surface of a Petri dish, and then immersed under culture medium in which cells are suspended. As chemokine molecules diffuse away from the spot, a transient chemoattractant gradient is established across the spots. Cells expressing the corresponding cognate chemokine receptors migrate against this gradient by crawling under the agarose spots towards their centre. We show that this migration is chemokine-specific; meaning that only cells that express the cognate chemokine cell surface receptor, migrate under the spot containing its corresponding chemokine ligand. Furthermore, we show that migration under the agarose spot can be modulated by selective small molecule antagonists present in the cell culture medium.

SUBMITTER: Ahmed M

PROVIDER: S-EPMC5430824 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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Agarose Spot as a Comparative Method for in situ Analysis of Simultaneous Chemotactic Responses to Multiple Chemokines.

Ahmed Mohaned M Basheer Haneen A HA Ayuso Jose M JM Ahmet Djevdet D Mazzini Marco M Patel Roshan R Shnyder Steven D SD Vinader Victoria V Afarinkia Kamyar K

Scientific reports 20170421 1

We describe a novel protocol to quantitatively and simultaneously compare the chemotactic responses of cells towards different chemokines. In this protocol, droplets of agarose gel containing different chemokines are applied onto the surface of a Petri dish, and then immersed under culture medium in which cells are suspended. As chemokine molecules diffuse away from the spot, a transient chemoattractant gradient is established across the spots. Cells expressing the corresponding cognate chemokin ...[more]

PMID: 28432337

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Project description:BackgroundThe role of chemokines in anaphylaxis is unclear.MethodsWe prospectively recruited 49 patients presenting to the emergency department with an acute episode of anaphylaxis and 28 healthy subjects. We measured serum levels of the chemokines CCL2, CCL5, CCL7, CCL8, CCL11, CCL13, CCL17, CCL21, CCL22, CCL24, and CCL26, tryptase, the absolute number of circulating basophils, monocytes, lymphocytes, and PMNs, and whole blood FCER1A, CPA3 and HDC gene expression at two time points: during the anaphylactic episode and in convalescent samples collected approximately 3 months later. We then investigated the in vitro chemotactic activity of chemokines induced during anaphylaxis for the in vitro migration of the corresponding cells.ResultsOnly CCL2 chemokine levels were significantly increased in anaphylaxis samples (median 514 pg/ml) compared to convalescent samples (284 pg/ml, P < 0.0001) and healthy subjects (279 pg/ml, P < 0.0001); there was no significant difference in any of the other chemokines. There was a significant positive correlation between the rates of increase of serum CCL2 (median [range]: 106.0% [- 44.7% to 557.4%]) and tryptase (133.8% [- 6.6% to 893.4%]; r = 0.68, P < 0.0001) and between the acute concentration of serum CCL2 and the acute concentration of serum tryptase (r = 0.77, P < 0.0001). The number of circulating basophils, but not other blood cells, significantly decreased during anaphylaxis (median 5.0 vs. 19.1 cells/µl in convalescent samples; P < 0.0001); a decrease in whole-blood gene expression of basophil markers (P ≤ 0.0018) confirmed these changes. Anaphylactic serum enhances the in vitro migration of basophils via CCL2-dependent chemotactic activity; in contrast, no CCL2-dependent chemotactic activity was observed for convalescent samples.ConclusionsOur findings imply an important and specific role for CCL2-mediated chemotactic activity in the pathophysiology of human anaphylaxis.

Dataset Information

Agarose Spot as a Comparative Method for in situ Analysis of Simultaneous Chemotactic Responses to Multiple Chemokines.

Publications

Agarose Spot as a Comparative Method for in situ Analysis of Simultaneous Chemotactic Responses to Multiple Chemokines.

Similar Datasets

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