The Experts below are selected from a list of 108 Experts worldwide ranked by ideXlab platform

Christian J Wiedermann - One of the best experts on this subject based on the ideXlab platform.

  • expression and function of the angiopoietin receptor tie 2 in human eosinophils
    The Journal of Allergy and Clinical Immunology, 2004
    Co-Authors: Clemens Feistritzer, Birgit A Mosheimer, Daniel H Sturn, Klaudija Bijuklic, Josef R Patsch, Christian J Wiedermann
    Abstract:

    Background Increased vascularity of bronchial mucosa is closely related to the expression of angiogenic factors, which contribute to the pathogenesis of diseases such as asthma bronchiale. Objective Here we examine the effects of the angiogenic growth factors angiopoietin 1 and angiopoietin 2 on eosinophil function in vitro and possible involvement of the angiopoietin receptor Tie-2. Methods Eosinophil migration was studied by Micropore Filter assays. Signaling mechanisms required for angiopoietin-dependent migration were tested by using signaling enzyme blockers. Tie-2 mRNA and receptor expression on the cell surface of eosinophils was demonstrated in RT-PCR and by fluorescence-activated cell sorting analysis. Results Angiopoietin 1 significantly stimulated eosinophil chemotaxis via activation of phosphodiesterase, phosphatidylinositol 3′-kinase, and tyrosine kinases. The effect on eosinophil migration of angiopoietin 1 was reversed by an antibody against the Tie-2 receptor and by angiopoietin 2. Incubation of eosinophils with angiopoietin 1 abolished the chemotactic effects of vascular endothelial growth factor on human eosinophils via the Tie-2 receptor. Finally, Tie-2 expression by human eosinophils was demonstrated on the transcriptional and protein level. Conclusions Data suggest that angiopoietin 1 stimulates directed migration and possibly inhibits vascular endothelial growth factor–induced eosinophil chemotaxis via its Tie-2 receptor, which is expressed by eosinophils. Thus, angiopoietin 1 may play an important role in the modulation of eosinophilic inflammation.

  • dendritic cell migration in different Micropore Filter assays
    Immunology Letters, 2000
    Co-Authors: Stefan Dunzendorfer, Arthur Kaser, Christian Meierhofer, Herbert Tilg, Christian J Wiedermann
    Abstract:

    Dendritic cells (DC) are highly motile and have been shown to migrate in vitro or in vivo towards various chemoattractants. Micropore Filter methods with polycarbonate Filters are generally used in these in vitro experiments. Among others, the main drawback of these Filters is their thickness, which does not allow any assessment of effects of absolute concentration compared to gradients. The aim of this study was to establish a chemotaxis assay for dendritic cells using nitrocellulose Filters, which can be adapted for checkerboard studies to distinguish between chemokinesis and chemotaxis. Immature DC were generated by culture of peripheral blood mononuclear cells using granulocyte-macrophage colony-stimulating factor and interleukin-4. We tested cell migration into nitrocellulose in a Boyden microchemotaxis chamber (leading front assay) and compared this method to the commonly used polycarbonate Filter technique. Dendritic cells migrated well into nitrocellulose towards gradients of formyl peptide, complement fragment 5a, and monocyte chemotactic protein-3. The nitrocellulose method appeared to be more sensitive as compared to experiments testing migration across polycarbonate Filters. Subsequent checkerboard analyses confirmed chemotactic activities of formyl peptide and complement fragment 5a. However, depending on the assay system, chemotaxis in polycarbonate Filters but chemokinesis in nitrocellulose Filters were observed for monocyte chemotactic protein-3. Measurement of DC migration in a cellulose nitrate Micropore Filter assay is more sensitive than the commonly used polycarbonate method and can be adapted for checkerboard analyses.

  • use of fluorochromes in the determination of chemotaxis and haptotaxis of granulocytes by Micropore Filter assays
    Annals of Hematology, 1996
    Co-Authors: Peter Schratzberger, Christian M Kahler, Christian J Wiedermann
    Abstract:

    Most conventional assays for the in vitro measurement of polymorphonuclear leukocyte (PMN) migration are modifications of the Boyden chamber technique, which requires quantification of migrated cells on Micropore Filters. This quantification is accompanied by several disadvantages, comprising errors in direct cell counting, low sensitivity of turbidimetric methods, the loss of marker enzymes by activation of PMNs, or the use of radioactive isotopes. We set up an improved fluorometric method to measure chemotactic and haptotactic migration of PMNs using polycarbonate Filter-bearing Transwell culture plate inserts. This improved fluorometric method allows the evaluation of effects of fluorescent-dye labeling on chemotaxis and haptotaxis - defined as cell migration due to cell surface-or matrix-bound gradients of chemoattractants —by collecting data in an automated system. Results were compared with data obtained from direct microscopic cell counting in Transwell experiments as well as in conventional 48-multiwell Boyden chamber assays. Calcein-AM and BCECF-AM proved to be fluorochromes with minimal effect on both types of PMN migration. We conclude that the fluorochromes are powerful tools for the analysis of PMN migration and allow modifications of chemotaxis/haptotaxis assays for automated quantification.

Peter Schratzberger - One of the best experts on this subject based on the ideXlab platform.

  • use of fluorochromes in the determination of chemotaxis and haptotaxis of granulocytes by Micropore Filter assays
    Annals of Hematology, 1996
    Co-Authors: Peter Schratzberger, Christian M Kahler, Christian J Wiedermann
    Abstract:

    Most conventional assays for the in vitro measurement of polymorphonuclear leukocyte (PMN) migration are modifications of the Boyden chamber technique, which requires quantification of migrated cells on Micropore Filters. This quantification is accompanied by several disadvantages, comprising errors in direct cell counting, low sensitivity of turbidimetric methods, the loss of marker enzymes by activation of PMNs, or the use of radioactive isotopes. We set up an improved fluorometric method to measure chemotactic and haptotactic migration of PMNs using polycarbonate Filter-bearing Transwell culture plate inserts. This improved fluorometric method allows the evaluation of effects of fluorescent-dye labeling on chemotaxis and haptotaxis - defined as cell migration due to cell surface-or matrix-bound gradients of chemoattractants —by collecting data in an automated system. Results were compared with data obtained from direct microscopic cell counting in Transwell experiments as well as in conventional 48-multiwell Boyden chamber assays. Calcein-AM and BCECF-AM proved to be fluorochromes with minimal effect on both types of PMN migration. We conclude that the fluorochromes are powerful tools for the analysis of PMN migration and allow modifications of chemotaxis/haptotaxis assays for automated quantification.

Stefan Dunzendorfer - One of the best experts on this subject based on the ideXlab platform.

  • dendritic cell migration in different Micropore Filter assays
    Immunology Letters, 2000
    Co-Authors: Stefan Dunzendorfer, Arthur Kaser, Christian Meierhofer, Herbert Tilg, Christian J Wiedermann
    Abstract:

    Dendritic cells (DC) are highly motile and have been shown to migrate in vitro or in vivo towards various chemoattractants. Micropore Filter methods with polycarbonate Filters are generally used in these in vitro experiments. Among others, the main drawback of these Filters is their thickness, which does not allow any assessment of effects of absolute concentration compared to gradients. The aim of this study was to establish a chemotaxis assay for dendritic cells using nitrocellulose Filters, which can be adapted for checkerboard studies to distinguish between chemokinesis and chemotaxis. Immature DC were generated by culture of peripheral blood mononuclear cells using granulocyte-macrophage colony-stimulating factor and interleukin-4. We tested cell migration into nitrocellulose in a Boyden microchemotaxis chamber (leading front assay) and compared this method to the commonly used polycarbonate Filter technique. Dendritic cells migrated well into nitrocellulose towards gradients of formyl peptide, complement fragment 5a, and monocyte chemotactic protein-3. The nitrocellulose method appeared to be more sensitive as compared to experiments testing migration across polycarbonate Filters. Subsequent checkerboard analyses confirmed chemotactic activities of formyl peptide and complement fragment 5a. However, depending on the assay system, chemotaxis in polycarbonate Filters but chemokinesis in nitrocellulose Filters were observed for monocyte chemotactic protein-3. Measurement of DC migration in a cellulose nitrate Micropore Filter assay is more sensitive than the commonly used polycarbonate method and can be adapted for checkerboard analyses.

Christian M Kahler - One of the best experts on this subject based on the ideXlab platform.

  • use of fluorochromes in the determination of chemotaxis and haptotaxis of granulocytes by Micropore Filter assays
    Annals of Hematology, 1996
    Co-Authors: Peter Schratzberger, Christian M Kahler, Christian J Wiedermann
    Abstract:

    Most conventional assays for the in vitro measurement of polymorphonuclear leukocyte (PMN) migration are modifications of the Boyden chamber technique, which requires quantification of migrated cells on Micropore Filters. This quantification is accompanied by several disadvantages, comprising errors in direct cell counting, low sensitivity of turbidimetric methods, the loss of marker enzymes by activation of PMNs, or the use of radioactive isotopes. We set up an improved fluorometric method to measure chemotactic and haptotactic migration of PMNs using polycarbonate Filter-bearing Transwell culture plate inserts. This improved fluorometric method allows the evaluation of effects of fluorescent-dye labeling on chemotaxis and haptotaxis - defined as cell migration due to cell surface-or matrix-bound gradients of chemoattractants —by collecting data in an automated system. Results were compared with data obtained from direct microscopic cell counting in Transwell experiments as well as in conventional 48-multiwell Boyden chamber assays. Calcein-AM and BCECF-AM proved to be fluorochromes with minimal effect on both types of PMN migration. We conclude that the fluorochromes are powerful tools for the analysis of PMN migration and allow modifications of chemotaxis/haptotaxis assays for automated quantification.

Altaf A. Wani - One of the best experts on this subject based on the ideXlab platform.

  • ATM recruitment and H2AX phosphorylation at damage sites is affected in XP cells during G1.
    2016
    Co-Authors: Alo Ray, Chessica Blevins, Gulzar Wani, Altaf A. Wani
    Abstract:

    (A) γH2AX foci formation at the CPD damage sites. OSU-2, XP-E, XP-C and XP-A cells were arrested in G1 by serum starvation for 48 h and exposed to 100 J/m2 UV irradiation using a 5 μm Micropore Filter. After 1 h post-exposure time immunofluorescence was performed as in Fig 5. The quantitative data of γH2AX and pATM foci formation were determined as described in Fig 2B. (B) pATM foci formation at the CPD damage sites. Experiments were done as described in A.

  • ATR and ATM recruitment and γH2AX phosphorylation at UVR damage sites in asynchronous OSU-2 cells.
    2016
    Co-Authors: Alo Ray, Chessica Blevins, Gulzar Wani, Altaf A. Wani
    Abstract:

    OSU-2 cells were exposed to 100 J/m2 UVR through a 5 μM Micropore Filter and allowed to respond to the induced damage for short indicated periods. The cells were fixed and immunofluorescence was performed to decorate ATR, pATM, γH2AX, and XPC proteins with cognate antibodies. Images show representative ATR, pATM and γH2AX foci co-localizing at DNA damage sites that were marked by distinct XPC labeling. (A) ATR (B) pATM (C) γH2AX (D) γH2AX foci formation in XP-E, XP-C, and XP-A cells upon 20 min post-repair.

  • ATM recruitment and H2AX phosphorylation at damage sites is not affected in XP cells during S.
    2016
    Co-Authors: Alo Ray, Chessica Blevins, Gulzar Wani, Altaf A. Wani
    Abstract:

    (A) γH2AX foci formation in EdU positive cells. Asynchronously growing OSU-2, XP-E, XP-C and XP-A cells were exposed to 100 J/m2 UVR through a 5 μm Micropore Filter. S phase cells were differentiated from non-S phase cells by nuclear EdU labeling as described in methods. The H2AX was detected by immunofluorescence as described in Fig 1. The quantitative data of γH2AX and pATM foci formation in S phase EdU-positive cells were determined as described in Fig 2B. (B) pATM foci formation in EdU positive cells. Experiments were done as described in A.

  • ATR, ATM recruitment and H2AX phosphorylation is affected in XP cells during G1.
    2016
    Co-Authors: Alo Ray, Chessica Blevins, Gulzar Wani, Altaf A. Wani
    Abstract:

    (A) Colocalization of ATR with DNA damage marker γH2AX in G1 arrested cells. OSU-2, XP-E, XP-C and XP-A cells were arrested in G1 phase of cell cycle by serum starvation for 48 h and exposed to 100 J/m2 UV irradiation through a 5 μm Micropore Filter. At 1 h post-exposure immunofluorescence was performed to determine the extent of co-localization. The quantitative data of ATR and γH2AX foci formation were determined as described in Fig 2B (B) Colocalization of pATM with γH2AX in G1 arrested cells. Experiments were done as described in A.