mouse anti human icam 1 pe  (Becton Dickinson)

Journal: Antioxidants

Article Title: Increased Adhesiveness of Blood Cells Induced by Mercury Chloride: Protective Effect of Hydroxytyrosol

doi: 10.3390/antiox13121576

Figure Lengend Snippet: Effect of HgCl 2 on the expression of endothelial adhesion molecules. HUVECs (Human Umbilical Vein Endothelial cells were treated) (24 h) with HgCl 2 (20 µM) or a vehicle and the expression of the adhesion molecules ICAM 1, VCAM-1, E-selectin, and P-selectin was measured by flow cytometry in HUVECs.

Article Snippet: BioLegend ® ), mouse anti-human CD62L PC7 (B30641, Lot: 200034, Beckman Coulter, San Diego, CA, USA), mouse anti-human ICAM-1 PE (555511, Lot: 0135026, BD Pharmingen™), mouse anti-human VCAM-1(CD106) FITC (551146, Lot: 2111025, BD Pharmingen™), mouse anti-human CD62P FITC (555523, Lot: 3191091, BD Pharmingen™), and mouse anti-human and CD62E PE (551145, Lot: 7180950, BD Pharmingen™).

Techniques: Expressing, Flow Cytometry

Journal: Mediators of Inflammation

Article Title: Tumour Cell Lines HT-29 and FaDu Produce Proinflammatory Cytokines and Activate Neutrophils In Vitro: Possible Applications for Neutrophil-Based Antitumour Treatment

doi: 10.1155/2009/817498

Figure Lengend Snippet: (a) Adhesion of PMN to HMVEC exposed to tumour cell conditioning medium. 2 × 10 6 DiI-labeled PMN were added to the culture wells containing confluent HMVEC previously treated overnight with none (control), HT-29 (HT-29) and FaDu (FaDu) 48 hours conditioning medium, and 10 U/mL of human recombinant IL-1 β (IL-1B) and then incubated at 37°C for 30 minutes. After washing, adhered PMN were evaluated as described in the Methods section. Bars represent the mean±SEM of 7 independent experiments performed in sixtoplicate. (b) Surface expression of ICAM-1 in HMVEC exposed to tumour cell conditioning media. HMVEC previously treated overnight with none (control), HT-29 (HT-29) and FaDu (FaDu) 48 hours conditioning medium, and 10 U/mL of human recombinant IL-1 β (IL-1B) were detached and analyzed for ICAM-1 expression by flow cytometry (see Methods). Upper panels represent histograms (out of 4) showing the effect of tumour cell conditioning medium on ICAM-1 expression. Bottom panels show quantitative data. Bars represent the mean ± SEM of 4 independent experiments. Statistical significance was assessed using ANOVA test; * P < .05, ** P < .01, and *** P < .001 when compared to control group.

Article Snippet: Twenty μ L of commercial solution of R-phycoerythrin (PE) labelled mouse anti-human ICAM-1 (CD54, ref 555511, BD Pharmingen) were then added and allowed to stand at 4°C in darkness for 20 minutes.

Techniques: Labeling, Recombinant, Incubation, Expressing, Flow Cytometry

Journal: Molecular Cancer

Article Title: E2F1 renders prostate cancer cell resistant to ICAM-1 mediated antitumor immunity by NF-κB modulation

doi: 10.1186/1476-4598-13-84

Figure Lengend Snippet: Expression of ICAM-1 is highly inversely correlated with E2F1 expression. A . ICAM-1 expression in DU145 cells. Whole cell lysates or total RNA were prepared from DU145 cells stably transfected with either the control vector or shRNA targeting E2F1. The expression of E2F1 and ICAM-1 was monitored by RT-PCR (upper) and Western blot (lower). B . ICAM-1 expression in DU145 cells was analyzed by real-time PCR. The stars indicate the significant differences ( P < 0.05). C . E2F1 overexpression plasmids or control were transient transfected to DU145 cells in a 6-well plate for 48 h. The expression of E2F1 and ICAM-1 were measured by RT-PCR. D . The expression of E2F1 and ICAM-1 was measured by real time PCR. The stars indicate the significant differences ( P < 0.05). E . Membrane expression of ICAM-1 in DU145/sh-E2F1 and control cells was measured by FACS. F . siRNA of E2F1 or scramble control was transient transfected to PC3 cells in a 6-well plate for 48 h. The expression of E2F1 and ICAM-1 in these cells were measured by RT-PCR. G . Forty pmol duplex siRNA of E2F1 or scramble control was transient transfected to Hela cells in a 6-well plate for 48 h. The expression of E2F1 and ICAM-1 in these cells were measured by RT-PCR (upper) and Western blotting analysis (lower).

Article Snippet: The following specific antibodies were used in this study: anti-p65, anti-p50, and anti-E2F1 polyclonal antibodies (Santa Cruz Biotech); anti-ICAM-1, anti-IκBα, anti-GAPDH, anti-phosphorylated p65 (p-Ser276), and anti-phosphorylated IkBα (p-Ser32/36) monoclonal antibodies (Cell Signaling); and PE mouse anti-human ICAM-1 antibody (BD Biosciences). pCMV-IκBαM plasmid was purchased from Clontech. pcDNA-E2F1 plasmid was a gift from Dr. Mian Wu.

Techniques: Expressing, Stable Transfection, Transfection, Plasmid Preparation, shRNA, Reverse Transcription Polymerase Chain Reaction, Western Blot, Real-time Polymerase Chain Reaction, Over Expression

Journal: Molecular Cancer

Article Title: E2F1 renders prostate cancer cell resistant to ICAM-1 mediated antitumor immunity by NF-κB modulation

doi: 10.1186/1476-4598-13-84

Figure Lengend Snippet: NF-κB binding sites are required for E2F1 regulation of ICAM-1 promoter. A . The histograph represent the human ICAM-1 promoter luciferase reporters used in this study. The location of each binding site on the promoter of ICAM-1 is indicated and labeled. The crossed boxes indicate the mutation sites on each construct. The mutated nucleotides corresponding to κB-1-mut and κB-2-mut are underlined. B and C . DU145/sh-Con and DU145/sh-E2F1 cells were co-transfected with duplex siRNA targeting p65 and either ICAM-1 wild type or mutant promoter reporters. The stars indicate the significant differences ( P < 0.05) (B) . The expression of membrane ICAM-1 was measured by Western blot (C) . The data are shown as the mean ± SD of triplicate measurements. D and E . DU145/sh-Con and DU145/sh-E2F1 cells were transiently transfected with either the empty vector or the mutant IκBα expressing plasmids. The phosphorylation of p65 and IκBα and the expression of ICAM-1 in these indicated cells were monitered by Western blot (D) . DU145/sh-Con and DU145/sh-E2F1 cells were co-transfected with IκBα expressing plasmids and either the ICAM-1 wild type or mutant promoter reporters for the luciferase activity assay (E) . The results are shown as the mean ± SD of triplicate measurements. The stars indicate the significant differences ( P < 0.05).

Article Snippet: The following specific antibodies were used in this study: anti-p65, anti-p50, and anti-E2F1 polyclonal antibodies (Santa Cruz Biotech); anti-ICAM-1, anti-IκBα, anti-GAPDH, anti-phosphorylated p65 (p-Ser276), and anti-phosphorylated IkBα (p-Ser32/36) monoclonal antibodies (Cell Signaling); and PE mouse anti-human ICAM-1 antibody (BD Biosciences). pCMV-IκBαM plasmid was purchased from Clontech. pcDNA-E2F1 plasmid was a gift from Dr. Mian Wu.

Techniques: Binding Assay, Luciferase, Labeling, Mutagenesis, Construct, Transfection, Expressing, Western Blot, Plasmid Preparation, Activity Assay

Journal: Molecular Cancer

Article Title: E2F1 renders prostate cancer cell resistant to ICAM-1 mediated antitumor immunity by NF-κB modulation

doi: 10.1186/1476-4598-13-84

Figure Lengend Snippet: RNA Silencing of E2F1 increases p65/p50 heterdimer binding to ICAM-1 promoter. A . ChIP assay was performed with cell lysates from DU145/sh-Con and DU145/sh-E2F1 cells. The chromatin was immunoprecipitated with anti-E2F1 and anti-p65 antibodies or normal IgG which served as a negative control. A pair of primers flanking the κB-1 binding site within the ICAM-1 promoter was used in PCR. PCR for the E2F1 binding site within the CDC2 promoter served as a positive control for detecting E2F1 binding activity. B . Real-time PCR was employed to the ChIP assay in (A). Relative occupancy values were calculated by determining the apparent immunoprecipitation efficiency (ratios of the amount of immunoprecipitated DNA to that of the input sample) and normalized to the level of p65 binding with ICAM-1 promoter in DU145/sh-Con cells, which was defined as 1.0. C . The relationship among E2F1 and p65/p50 heterodimer in DU145/sh-Con and DU145/sh-E2F1 cells was examined by coimmunoprecipitation analysis. Anti-p65 antibody was used for immunoprecipitation. The amounts of E2F1, p65 and p50 in the immunoprecipitates were detected by Western blot with the indicated specific antibodies.

Article Snippet: The following specific antibodies were used in this study: anti-p65, anti-p50, and anti-E2F1 polyclonal antibodies (Santa Cruz Biotech); anti-ICAM-1, anti-IκBα, anti-GAPDH, anti-phosphorylated p65 (p-Ser276), and anti-phosphorylated IkBα (p-Ser32/36) monoclonal antibodies (Cell Signaling); and PE mouse anti-human ICAM-1 antibody (BD Biosciences). pCMV-IκBαM plasmid was purchased from Clontech. pcDNA-E2F1 plasmid was a gift from Dr. Mian Wu.

Techniques: Binding Assay, Immunoprecipitation, Negative Control, Positive Control, Activity Assay, Real-time Polymerase Chain Reaction, Western Blot

Journal: Molecular Cancer

Article Title: E2F1 renders prostate cancer cell resistant to ICAM-1 mediated antitumor immunity by NF-κB modulation

doi: 10.1186/1476-4598-13-84

Figure Lengend Snippet: E2F1 knockdown enhances cell adhesion through ICAM-1. A . DU145/sh-Con and DU145/sh-E2F1 cells were pre-incubated with anti-ICAM-1 antibody or transient transfected with duplex siRNA of ICAM-1. PBMC labeled with Calcein-AM were added to the indicated cells. After washing three times, attached PBMC were captured using a fluorescence microscope at nine randomly selected fields of each well. B . The number of attached PBMC in the (A) was presented by the bar graph (white bar, DU145/sh-Con; gray bar, DU145/sh-E2F1). The data are shown as the mean value ± SD of triplicate measurement. The stars indicate the significant differences ( P < 0.05). C . RT-PCR was employed to validate the efficiency of the siRNA-ICAM-1 in DU145/sh-Con and DU145/sh-E2F1 cells that were used in (A) .

Article Snippet: The following specific antibodies were used in this study: anti-p65, anti-p50, and anti-E2F1 polyclonal antibodies (Santa Cruz Biotech); anti-ICAM-1, anti-IκBα, anti-GAPDH, anti-phosphorylated p65 (p-Ser276), and anti-phosphorylated IkBα (p-Ser32/36) monoclonal antibodies (Cell Signaling); and PE mouse anti-human ICAM-1 antibody (BD Biosciences). pCMV-IκBαM plasmid was purchased from Clontech. pcDNA-E2F1 plasmid was a gift from Dr. Mian Wu.

Techniques: Incubation, Transfection, Labeling, Fluorescence, Microscopy, Reverse Transcription Polymerase Chain Reaction

Journal: Molecular Cancer

Article Title: E2F1 renders prostate cancer cell resistant to ICAM-1 mediated antitumor immunity by NF-κB modulation

doi: 10.1186/1476-4598-13-84

Figure Lengend Snippet: RNA Silencing of E2F1 Sensitizes Prostate Cancer Cells to ICAM-1 Mediated Anti-tumor Responses. A . E2F1 knockdown increases cytotoxicity through ICAM-1. DU145/sh-Con and DU145/sh-E2F1 cells were transiently transfected with duplex siRNA of ICAM-1. The DU145 derived cells were used as the target cells and the CIK cells as the effector cells. Both of them at various ratios of effector cells to target cells were mixed and cytotoxicity was measured by 51 Cr release. The results are shown as the mean ± SD of triplicate measurements. B . The expression of membrane ICAM-1 in the indicated cells was measured by FACS with ICAM-1 specific antibody. C . The expression of IL-1β, TNF-α, IL-6 and IL-8 in DU145/sh-Con and DU145/sh-E2F1 cells were measured by real-time PCR. The data presents the fold-induction of the levels of each tested cytokine in DU145/sh-E2F1 cells over DU145/sh-Con cell, and represent the mean ± SD of triplicate measurements. D . E2F1 knockdown inhibits tumor xenografts growth in vivo, DU145 cells were stably transfected with the control vectors or shRNA expressing plasmids targeting E2F1 or ICAM-1 respectively and injected subcutaneously into nude mice. Tumor volumes were measured and estimated by the formula: length (mm) X width (mm) X height (mm)/2. The results are shown as the mean value ± SD of at least five tumors in each group. E and F . E2F1 knockdown increases ICAM-1-mediated leucocytes infiltration. The infiltrating leucocytes to DU145-derived tumors in nude mice were stained by H&E and indicated by arrows. The expression of E2F1 and ICAM-1 in these DU145-derived tumors was monitored by immunohistochemistry analysis, H&E staining (D) and Western blot (E) .

Article Snippet: The following specific antibodies were used in this study: anti-p65, anti-p50, and anti-E2F1 polyclonal antibodies (Santa Cruz Biotech); anti-ICAM-1, anti-IκBα, anti-GAPDH, anti-phosphorylated p65 (p-Ser276), and anti-phosphorylated IkBα (p-Ser32/36) monoclonal antibodies (Cell Signaling); and PE mouse anti-human ICAM-1 antibody (BD Biosciences). pCMV-IκBαM plasmid was purchased from Clontech. pcDNA-E2F1 plasmid was a gift from Dr. Mian Wu.

Techniques: Transfection, Derivative Assay, Expressing, Real-time Polymerase Chain Reaction, In Vivo, Stable Transfection, shRNA, Injection, Staining, Immunohistochemistry, Western Blot

Journal: Molecular Cancer

Article Title: E2F1 renders prostate cancer cell resistant to ICAM-1 mediated antitumor immunity by NF-κB modulation

doi: 10.1186/1476-4598-13-84

Figure Lengend Snippet: A schematic model whereby E2F1 regulates the ICAM-1 mediated anti-tumor immune circuit through NF-κB modulation. NF-κB binding site is required for E2F1 regulation of ICAM-1. E2F1 interacts with NF-κB forming an NF-κB/E2F1 complex. E2F1 acts as a suppressor to prevent the NF-κB p65/p50 complex from binding to ICAM-1 promoter. As a consequence, E2F1 interferes with the adhesion of monocytes onto prostate cancer cells, the sensitivity of tumor cells to the cytotoxic effect of cytokine-induced killer cells and the growth of prostate cancer cells. Targeted knockdown of E2F1 does not affect the expression of phosphorylation of NF-κB p65 and IκBα, but releases NF-κB p65 to facilitate p65/p50 heterodimerization and binding to the ICAM-1 promoter. Subsequently, ICAM-1 transcription and production are induced, resulting in the enhanced immune cells mediated cytotoxicity against prostate carcinoma cells.

Article Snippet: The following specific antibodies were used in this study: anti-p65, anti-p50, and anti-E2F1 polyclonal antibodies (Santa Cruz Biotech); anti-ICAM-1, anti-IκBα, anti-GAPDH, anti-phosphorylated p65 (p-Ser276), and anti-phosphorylated IkBα (p-Ser32/36) monoclonal antibodies (Cell Signaling); and PE mouse anti-human ICAM-1 antibody (BD Biosciences). pCMV-IκBαM plasmid was purchased from Clontech. pcDNA-E2F1 plasmid was a gift from Dr. Mian Wu.

Techniques: Binding Assay, Expressing

Journal: PLoS ONE

Article Title: Hydrodynamic Regulation of Monocyte Inflammatory Response to an Intracellular Pathogen

doi: 10.1371/journal.pone.0014492

Figure Lengend Snippet: (A) THP-1 monocytes were infected with mock PBS or chlamydial EB (MOI 2) for 16 h. Uninfected and infected cells were sheared for 20 min at 0 (static) or 10 (shear) dyn/cm 2 using a cone-and-plate viscometer, and were incubated for either 30 min or 3 h post shear. The cells were fixed, stained with FITC-conjugated ICAM-1 antibody and analyzed by flow cytometry. The * denote statistically significant increase ( P <0.05, Student's t -test) in ICAM-1 expression due to shear; (B) HAECs were activated with 100 nM TNF-α and assembled in a parallel plate flow chamber. Infected monocytes were sheared either at 0 (static) or 10 (shear) dyn/cm 2 for 20 min, incubated for 30 min, and then perfused through the flow chamber at 1 dyn/cm 2 . The number of adherent cells was counted from four different fields of view. The results are mean ± SD of one representative experiment performed in triplicate, and the experiments were repeated three times. The *, **, and *** denote statistically significant increase in the number of infected, monocytes adhered to HAEC between different groups ( P <0.05, ANOVA).

Article Snippet: After blocking in 2% BSA in PBS for 30 minutes, the cells were washed once with PBS and incubated with 10 µl of PE-conjugated mouse monoclonal anti-human ICAM-1 antibody per 10 6 cells (eBiosciences) for 1 h. After staining, the cells were washed three times with PBS, and resuspended in 500 µl PBS.

Techniques: Infection, Incubation, Staining, Flow Cytometry, Expressing

Journal: Epigenomes

Article Title: Epigenetic Immune Remodeling of Mesothelioma Cells: A New Strategy to Improve the Efficacy of Immunotherapy

doi: 10.3390/epigenomes5040027

Figure Lengend Snippet: Flow cytometry analysis of changes induced by different epigenetic drugs in HLA class I and ICAM-1 molecules expression. MPM cell lines untreated or treated with guadecitabine, VPA, SAHA, EPZ-6438, or guadecitabine-based combinations were incubated with ( A ) anti-human HLA class I or ( B ) anti-human ICAM-1 mAbs and studied by flow cytometry. Data obtained were analyzed by Kaluza software. Values reported correspond to MFI in treated vs. untreated cells. Each data point represents mean value of MFI obtained in 3 independent experiments for each single cell line.

Article Snippet: PE mouse anti-human ICAM-1 clone 84H10 monoclonal antibody (mAb) was purchased from Beckman Coulter and alexafluor 488 mouse anti-human HLA-ABC (class I) clone W6/32 mAb was purchased from Biolegend.

Techniques: Flow Cytometry, Expressing, Incubation, Software