Journal: Allergy, Asthma & Immunology Research

Article Title: Enhanced Allergic Inflammation of Der p 2 Affected by Polymorphisms of MD-2 Promoter

doi: 10.4168/aair.2015.7.5.497

Figure Lengend Snippet: Differences in cytokine secretion with and without Der p2 and LPS

Article Snippet: Cells were fixed with cytofix/cytoperm at room temperature for 30 minutes, washed with PBS, and stained with fluorescein isothiocyanate-conjugated rat anti-human IFN-γ monoclonal antibody (BD Biosciences) and R-phycoerythrin-conjugated rat anti-human IL-4 monoclonal antibody (BD Biosciences) at room temperature for 30 minutes and washed with PBS.

Techniques:

Journal: Allergy, Asthma & Immunology Research

Article Title: Enhanced Allergic Inflammation of Der p 2 Affected by Polymorphisms of MD-2 Promoter

doi: 10.4168/aair.2015.7.5.497

Figure Lengend Snippet: MD-2 promoter SNPs (rs1809441/rs1809442) affect the binding affinity of GATA-3 and T H 2-cytokine expression. The binding activity of GATA-3 and T H 2-cytokine expression were analyzed by electrophoretic mobility shift assay (EMSA) and flow cytometry, respectively. The human promyelocytic leukemia cell line (HL-60) and human monocyte derived from donor' PBMCs were employed for GATA-3 binding activity. The binding activity of GATA-3 to these MD-2 promoter SNPs was analyzed by EMSA using an oligonucleotide containing the tandem GATA motif (-136 to -161). (A) The binding activity of the transcription factor GATA-3 was induced by Der p 2 (1.5 µg/mL) at different time points (0-8 hours) with the human promyelocytic leukemia cell line HL-60. (B) Human monocytes derived from subjects with (+) or without (-) MD-2 promoter SNPs were collected to analyze the binding affinity of GATA-3 . SNP (+), MD-2 promoter SNPs (rs1809441-T/rs1809442-G); SNP (-), MD-2 promoter SNPs (rs1809441-G /rs1809442-C). Quantification: hybridization signals were quantified by the image analysis program. (C) Cytokines in activated T H cells derived from donor's leukocytes underwent immunofluorescence staining and flow cytometry. Three-color staining methods were used to analyze the expressions of IL-4 and interferon (IFN)-γ in CD4+ cells. Mean fluorescence was measured using a flow cytometer and is presented in percentages. A total of 5,000 cells were analyzed in each sample.

Article Snippet: Cells were fixed with cytofix/cytoperm at room temperature for 30 minutes, washed with PBS, and stained with fluorescein isothiocyanate-conjugated rat anti-human IFN-γ monoclonal antibody (BD Biosciences) and R-phycoerythrin-conjugated rat anti-human IL-4 monoclonal antibody (BD Biosciences) at room temperature for 30 minutes and washed with PBS.

Techniques: Binding Assay, Expressing, Activity Assay, Electrophoretic Mobility Shift Assay, Flow Cytometry, Derivative Assay, Hybridization, Immunofluorescence, Staining, Fluorescence

Journal: Journal of Inflammation (London, England)

Article Title: Colocalization of endogenous TNF with a functional intracellular splice form of human TNF receptor type 2

doi: 10.1186/1476-9255-2-7

Figure Lengend Snippet: Colocalization of hicp75TNFR with endogenous or exogenous TNF. (A) L929 cells were transduced with hicp75TNFR-YFP and exposed to biotinylated TNF. Receptosomes were isolated after different times, subjected to SDS-PAGE and analyzed by immunostaining of blotted proteins. (B) L929 cells expressing hTNF were transduced with hicp75TNFR and stained with rabbit anti-human TNF antibodies and mouse monoclonal antibody anti-human p75TNFR (80M2).

Article Snippet: Expression of transduced hp75TNFR isoforms on the cell surface was detected by flow cytometry on a FACStar Plus (Becton Dickinson, San Jose, CA, USA) using the PE-coupled specific rat anti-human p75TNFR monoclonal antibody and PE-labelled rat IgG2b (both BD, Heidelberg, Germany) as isotype control.

Techniques: Transduction, Isolation, SDS Page, Immunostaining, Expressing, Staining