fitc conjugated mouse monoclonal anti human e selectin antibody  (R&D Systems)

Journal: Frontiers in Cell and Developmental Biology

Article Title: CRISPR-Based Activation of Endogenous Expression of TPM1 Inhibits Inflammatory Response of Primary Human Coronary Artery Endothelial and Smooth Muscle Cells Induced by Recombinant Human Tumor Necrosis Factor α

doi: 10.3389/fcell.2021.668032

Figure Lengend Snippet: Effect of TNFα on activation of pHCAECs. (A) Representative micrographs of intravital detection of E-selectin using magnetic beads coated with anti-E-selectin monoclonal antibodies (bright field). F-actin (red) and nuclei (blue) were stained fluorescently after fixation. CTRL refers to the control (top panel), TNFα 100 ng/ml to cells treated with rh TNFα (bottom panel), mAb to monoclonal antibodies. (B) Representative micrographs of intravital co-culture of pHCAECs (contrast phase) with Jurkat T cells (contrast phase and fluorescence). Nuclei of Jurkat T cells (blue) were stained with Hoechst 33342. CTRL refers to the control (top panel), TNFα 100 ng/ml to cells treated with rh TNFα (bottom panel). (C) Number of adherent Jurkat T cells to pHCAECs per microscopic field from the control and TNFα-activated pHCAECs after 30 min of their co-culture. CTRL refers to the control (top panel), TNFα 100 ng/ml to cells treated with rh TNFα, **** to p < 0.0001, as determined by unpaired t -test. (D) Velocity of motile Jurkat T cells on pHCAECs during 30 min of their co-culture. CTRL refers to the control (top panel), TNFα 100 ng/ml to cells treated with rh TNFα. **** p < 0.0001, as determined by unpaired t -test.

Article Snippet: The membrane was then blocked for 10 min in a SuperSignal Western Blot Enhancer (Thermo Fisher Scientific) and incubated for 2.5 h or overnight in an iBind Flex Solution (Thermo Fisher Scientific) with (i) mouse anti-claudin-5 (clone: 4C3C2) monoclonal antibody (1:500) and goat anti-mouse–horseradish peroxidase secondary antibody (HRP, 1:2,000; both from Thermo Fisher Scientific); (ii) mouse anti-GAPDH (clone: ZG003) monoclonal antibody (1:500) and goat anti-mouse–HRP secondary antibody (1:2,000; both from Thermo Fisher Scientific); (iii) mouse anti-α-tropomyosin (clone: F-6) monoclonal antibody (1:100, Santa Cruz) and goat anti-mouse-HRP secondary antibody (1:2,000, Thermo Fisher Scientific); (iv) mouse anti-E-selectin (clone: CL2/6) monoclonal antibody (1:100) and goat anti-mouse–HRP secondary antibody (1:2,000; both from Thermo Fisher Scientific); (v) mouse anti-talin (clone: 8D4) monoclonal antibody (1:100, Sigma-Aldrich) and goat anti-mouse–HRP secondary antibody (1:2,000, Thermo Fisher Scientific); (vi) mouse anti-vinculin (clone: J144) monoclonal antibody (1:1,000) and goat anti-mouse-HRP secondary antibody (1:2,000; both from Thermo Fisher Scientific); (vii) mouse anti-α-catenin (clone: 7A4) monoclonal antibody (1:250) and goat anti-mouse–HRP secondary antibody (1:2,000; both from Thermo Fisher Scientific); (viii) mouse anti-β-catenin (clone: 5H10) monoclonal antibody (1:250) and goat anti-mouse–HRP secondary antibody (1:2,000; both from Thermo Fisher Scientific); (iv) rabbit anti-VE-cadherin polyclonal antibody (1:500) and goat anti-mouse–HRP secondary antibody (1:2,000; both from Thermo Fisher Scientific) using the iBind Flex Western System (Thermo Fisher Scientific).

Techniques: Activation Assay, Magnetic Beads, Staining, Co-Culture Assay, Fluorescence

Journal: Frontiers in Cell and Developmental Biology

Article Title: CRISPR-Based Activation of Endogenous Expression of TPM1 Inhibits Inflammatory Response of Primary Human Coronary Artery Endothelial and Smooth Muscle Cells Induced by Recombinant Human Tumor Necrosis Factor α

doi: 10.3389/fcell.2021.668032

Figure Lengend Snippet: Effect of TNFα on posttranslational expression of cell–cell and cell–ECM junctional proteins in pHCAECs with CRISPR-based modulated expression of TPM1 . (A) Schematic overview of CRISPR systems used in regulation of TPM1 expression in pHCAECs. The cells were nucleofected with a CRISPR control plasmid encoding the Cas9 nuclease and a non-specific guide RNA (upper left panel), CRISPR TPM1 activation system encoding the nuclease-deficient dCas9 fused to the transactivation domain VP64, a plasmid encoding the MS2-p65-HSF1 fusion protein, and a guide RNA targeting sequences upstream of the TPM1 transcriptional start site (upper left panel) or CRISPR TPM1 -knockout system encoding the Cas9 nuclease and a TPM1 -specific 20-nt guide RNA (lower panel). (B) Representative micrograph of intravital detection of pmaxGFP plasmid product (green fluorescence) in nucleofected pHCAECs (contrast phase). (C) Representative western blots, surface plot, and densitometric analysis for α-tropomyosin (upper panel) and GAPDH (lower panel). CTRL refers to the control, ** to p < 0.01, **** to p < 0.0001. (D) Representative western blots, surface plot, and densitometric analysis for E-selectin. CTRL refers to the control (upper panel), TNFα 100 ng/ml to cells treated with rh TNFα (lower panel), NS to non-significant, * to p < 0.05, ** to p < 0.01, **** p < 0.0001, as determined by the Kruskal–Wallis test, $$ to p < 0.01, as determined by unpaired t -test. (E) Representative western blots, surface plot, and densitometric analysis for talin. CTRL refers to the control (upper panel), TNFα 100 ng/ml to cells treated with rh TNFα (lower panel), NS to non-significant, ** to p < 0.01, *** to p < 0.001, **** to p < 0.0001, as determined by the Kruskal–Wallis test, $$ to p < 0.01, $$$ to p < 0.001, $$$$ p < 0.0001, as determined by unpaired t -test. (F) Representative western blots, surface plot, and densitometric analysis for vinculin. CTRL refers to the control (upper panel), TNFα 100 ng/ml to cells treated with rh TNFα (lower panel), NS to non-significant, * to p < 0.05, ** to p < 0.01, as determined by the Kruskal–Wallis test, $$ to p < 0.01, as determined by unpaired t -test. (G) Representative western blots, surface plot, and densitometric analysis for α-catenin. CTRL refers to the control (upper panel), TNFα 100 ng/ml to cells treated with rh TNFα (lower panel), NS to non-significant, * to p < 0.05, ** to p < 0.01, *** to p < 0.001, as determined by the Kruskal–Wallis test, $ to p < 0.05, as determined by unpaired t -test. (H) Representative western blots, surface plot, and densitometric analysis for β-catenin. CTRL refers to the control (upper panel), TNFα 100 ng/ml to cells treated with rh TNFα (lower panel), NS to non-significant, * to p < 0.05, **** to p < 0.0001, as determined by the Kruskal–Wallis test, $$$ to p < 0.001, as determined by unpaired t -test. (I) Representative western blots, surface plot, and densitometric analysis for VE-cadherin. CTRL refers to the control (upper panel), TNFα 100 ng/ml to cells treated with rh TNFα (lower panel), NS to non-significant, ** to p < 0.01, *** to p < 0.001, **** to p < 0.0001, as determined by the Kruskal–Wallis test, $$ to p < 0.01, as determined by unpaired t -test.

Article Snippet: The membrane was then blocked for 10 min in a SuperSignal Western Blot Enhancer (Thermo Fisher Scientific) and incubated for 2.5 h or overnight in an iBind Flex Solution (Thermo Fisher Scientific) with (i) mouse anti-claudin-5 (clone: 4C3C2) monoclonal antibody (1:500) and goat anti-mouse–horseradish peroxidase secondary antibody (HRP, 1:2,000; both from Thermo Fisher Scientific); (ii) mouse anti-GAPDH (clone: ZG003) monoclonal antibody (1:500) and goat anti-mouse–HRP secondary antibody (1:2,000; both from Thermo Fisher Scientific); (iii) mouse anti-α-tropomyosin (clone: F-6) monoclonal antibody (1:100, Santa Cruz) and goat anti-mouse-HRP secondary antibody (1:2,000, Thermo Fisher Scientific); (iv) mouse anti-E-selectin (clone: CL2/6) monoclonal antibody (1:100) and goat anti-mouse–HRP secondary antibody (1:2,000; both from Thermo Fisher Scientific); (v) mouse anti-talin (clone: 8D4) monoclonal antibody (1:100, Sigma-Aldrich) and goat anti-mouse–HRP secondary antibody (1:2,000, Thermo Fisher Scientific); (vi) mouse anti-vinculin (clone: J144) monoclonal antibody (1:1,000) and goat anti-mouse-HRP secondary antibody (1:2,000; both from Thermo Fisher Scientific); (vii) mouse anti-α-catenin (clone: 7A4) monoclonal antibody (1:250) and goat anti-mouse–HRP secondary antibody (1:2,000; both from Thermo Fisher Scientific); (viii) mouse anti-β-catenin (clone: 5H10) monoclonal antibody (1:250) and goat anti-mouse–HRP secondary antibody (1:2,000; both from Thermo Fisher Scientific); (iv) rabbit anti-VE-cadherin polyclonal antibody (1:500) and goat anti-mouse–HRP secondary antibody (1:2,000; both from Thermo Fisher Scientific) using the iBind Flex Western System (Thermo Fisher Scientific).

Techniques: Expressing, CRISPR, Plasmid Preparation, Activation Assay, Knock-Out, Fluorescence, Western Blot

Journal: PLoS ONE

Article Title: Circulating Tumor Cells from Prostate Cancer Patients Interact with E-Selectin under Physiologic Blood Flow

doi: 10.1371/journal.pone.0085143

Figure Lengend Snippet: MDA cells were labeled with mAb J591-488 for PSMA. After labeling, 10 6 J591-488 labeled MDA cells were perfused at 0.5, 1, 3, 5, and 8 dyne/cm 2 shear stress. Similarly unlabeled MDA cells were also perfused through E-selectin coated microtubes. Ten videos were taken at different lengths of the microtube for each shear stress. Rolling velocity was measured for both unlabeled and anti-PSMA J591-488 labeled MDA cells. Figure shows no significant difference in the rolling velocity between unlabeled and anti-PSMA J591-488 labeled MDA cells at shear stress ranging from 0.5-1 dyn/cm 2 . The mean rolling velocities at 0.5 dyn/cm 2 were 5.27 + 1.38 and 5.23 + 1.85 µm/sec in unlabeled and J591-488 labeled MDA cells, respectively. At higher shear stresses, a significant difference was observed between the two categories of MDA cells. Histogram shows the results from three separate experiments combined together. UnL= unlabeled MDA cells, Lab= J591-488 labeled MDA cells. Data is represented as Mean + SD.

Article Snippet: For immunofluorescence, both 1° and E4ORF1 HUVECs were stimulated with IL-1β for 4h and both unstimulated and IL-1β-stimulated HUVECs were fixed, permeabilized with 0.1% Triton-X 100, and immunostained with mouse anti-E-selectin monoclonal antibody (2.5 µg/ml, 68-5H11, BD Pharmingen, San Jose, CA) overnight at 4°C.

Techniques: Labeling

Journal: PLoS ONE

Article Title: Circulating Tumor Cells from Prostate Cancer Patients Interact with E-Selectin under Physiologic Blood Flow

doi: 10.1371/journal.pone.0085143

Figure Lengend Snippet: Interactions between CTCs derived from metastatic PCa patients and E-selectin-coated microtube surfaces.

Article Snippet: For immunofluorescence, both 1° and E4ORF1 HUVECs were stimulated with IL-1β for 4h and both unstimulated and IL-1β-stimulated HUVECs were fixed, permeabilized with 0.1% Triton-X 100, and immunostained with mouse anti-E-selectin monoclonal antibody (2.5 µg/ml, 68-5H11, BD Pharmingen, San Jose, CA) overnight at 4°C.

Techniques: Derivative Assay

Journal: PLoS ONE

Article Title: Circulating Tumor Cells from Prostate Cancer Patients Interact with E-Selectin under Physiologic Blood Flow

doi: 10.1371/journal.pone.0085143

Figure Lengend Snippet: A ) Western blot showing E-selectin protein expression in primary (1°) and E4ORF1 HUVECs. HUVECs were either stimulated with 50 ng/ml IL-1β for 4 h or left untreated. E-selectin= 95-115 kDa, based on post-translational modifications. The blot underneath E-selectin shows actin, used as a loading control. Western blot is a representative of three independent experiments. B ) Immunofluorescence showing E-selectin expression in IL-1β-stimulated 1° HUVECs (passage=2) and E4ORF1 HUVECs (passage 10). E-selectin= Green, DAPI= Blue. C ) Rolling velocity of 10 6 MDA cells on 50 ng/ml IL-1β-stimulated 1°- and E4ORF1- HUVECs. HUVECs were stimulated with IL-1β for 4 h. The mean rolling velocity of MDA cells at 1 dyn/cm 2 was 5.94 + 3.43 µm/sec and 6.35 + 3.92 µm/sec on E4-ORF1 and primary HUVECs, respectively. No significant difference was seen in the rolling velocities of MDA cells on either 1° or E4ORF1- HUVECs. Graph depicts Mean + SD.

Article Snippet: For immunofluorescence, both 1° and E4ORF1 HUVECs were stimulated with IL-1β for 4h and both unstimulated and IL-1β-stimulated HUVECs were fixed, permeabilized with 0.1% Triton-X 100, and immunostained with mouse anti-E-selectin monoclonal antibody (2.5 µg/ml, 68-5H11, BD Pharmingen, San Jose, CA) overnight at 4°C.

Techniques: Western Blot, Expressing, Immunofluorescence

Journal: PLoS ONE

Article Title: Circulating Tumor Cells from Prostate Cancer Patients Interact with E-Selectin under Physiologic Blood Flow

doi: 10.1371/journal.pone.0085143

Figure Lengend Snippet: 1X10 6 MDA cells were perfused over HUVECs. HUVECs used in the experiment were: a ) stimulated with 50 ng/ml IL-1β for 4 h, b ) unstimulated, and c ) stimulated with 50 ng/ml IL-1β for 4h plus 1 h anti-E-selectin neutralizing antibody. Box plot shows the rolling velocities of MDA cells in three different categories of HUVECs. The rolling velocities in IL-1β-stimulated HUVECs ranged from 3.72 + 2.1 to 6.01 +2.45 µm/sec at different shear stresses. The dots represent the outliers. No box plot for the category means that no cells were found rolling at the mentioned shear stress. Box plots represent three separate experiments combined together. n= total number of cells rolling in 10 different fields at a given shear stress in three independent experiments. Notice the reduction in the number of cells (n) rolling in StNeu and Unst versus St category. St= IL-1β-stimulated HUVECs expressing E-selectin. StNeu= IL-1β-stimulated HUVECs incubated with anti-E-selectin neutralizing antibody. Unst= E4ORF1 HUVECs. p < 0.05 was considered significant.

Article Snippet: For immunofluorescence, both 1° and E4ORF1 HUVECs were stimulated with IL-1β for 4h and both unstimulated and IL-1β-stimulated HUVECs were fixed, permeabilized with 0.1% Triton-X 100, and immunostained with mouse anti-E-selectin monoclonal antibody (2.5 µg/ml, 68-5H11, BD Pharmingen, San Jose, CA) overnight at 4°C.

Techniques: Expressing, Incubation

Journal: Frontiers in Aging Neuroscience

Article Title: A Comorbid Rat Model of Neuroendocrine-Immune System Alterations Under the Impact of Risk Factors for Stroke

doi: 10.3389/fnagi.2021.827503

Figure Lengend Snippet: Changes in expression levels of ICAM-1, VCAM-1, and E-selectin in the cerebral microvasculature in rats. (A) Light photomicrograph of the isolated microvascular fraction from rat cerebral cortex. Scale bar = 100 μm. (B–E) Representative western blot of ICAM-1, VCAM-1, and E-selectin in cerebral microvessels. Quantification of ICAM-1, VCAM-1, and E-selectin normalized with β-actin. RD, control group; HCD, high cholesterol diet group; HCD + CUR, high cholesterol diet receiving a daily dose of 200 mg/kg body weight curcumin by gavage for 4 weeks. Carotid injury was induced by an air-drying endothelial denudation method at the end of the second week. Data are representative of two independent experiments and are expressed as mean ± SE, n = 4 for each group. * P < 0.05, ** P < 0.01.

Article Snippet: Total protein samples extracted from the dissected hypothalamic tissue and the microvessel fraction were subjected to electrophoresis, transferred onto PVDF membranes, and incubated with primary antibodies comprising: mouse anti-neuropeptide Y (NPY) monoclonal antibodies (Santa Cruz Biotechnology, Inc., United States), rabbit anti-VCAM-1 polyclonal antibody (Cell Signaling Technology, United States), rabbit anti-ICAM-1 polyclonal antibody (Cell Signaling Technology, United States), and mouse anti-E-selectin monoclonal antibody (Santa Cruz Biotechnology, Inc., United States). β-Actin antibody was used for sample normalization to express the final result as the intensity ratio (% sham-operated control).

Techniques: Expressing, Isolation, Western Blot, Control

Journal: Nutrients

Article Title: Co-toxicity of Endotoxin and Indoxyl Sulfate, Gut-Derived Bacterial Metabolites, to Vascular Endothelial Cells in Coronary Arterial Disease Accompanied by Gut Dysbiosis

doi: 10.3390/nu14030424

Figure Lengend Snippet: E-selectin and MCP-1 production in HUVECs stimulated with LPS and indoxyl. ( a ) E-selectin levels in HUVECs treated directly (blue bars) with LPS and IS or both metabolites and indirectly (green bars) with CM25-LPS/CM25-IS at appropriate concentrations. The asterisks above bars indicate the statistical differences in E-selectin levels between HUVECs stimulated directly with LPS/IS or indirectly with CM25. E-selectin was evaluated in HUVECs stimulated with LPS or IS or both bacterial metabolites for 18 h with FITC-conjugated mouse monoclonal anti-human E-selectin antibody. Data are presented as means ± SEM from two independent assays performed in quadruplicate. ( b ) MPC-1 levels in CM25 from MDM (black bars) and a culture media from HUVECs stimulated for 18 h with CM25 (green bars) or with LPS/IS (blue bars). The asterisks above black bars represent statistically significant differences between MCP-1 levels in CM25 and a culture media from HUVECs stimulated with LPS and IS for 18 h. The asterisks above white and grey bars indicate differences between MCP-1 levels in a culture media from HUVECs stimulated with CM25 and LPS/IS. MCP-1 was evaluated using the MCP-1 Human ELISA kit. Data are the means ± SEM from two independent assays performed in duplicate.

Article Snippet: Briefly, confluent HUVECs layers in a 6-well plate stimulated with CM25 or LPS and IS for 18 h was washed twice with PBS and fixed with cold 0.5% formalin in PBS for 2.5 min. Then, cells were washed with HBSS-BSA (0.5% w / v ) before adding FITC-conjugated mouse monoclonal anti-human E-selectin antibody (R&D Systems, Abingdon, United Kingdom) at concentration 10 μg per 1 × 10 6 cells in HBSS with 0.02% saponin ( w / v ).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Aging (Albany NY)

Article Title: Supplementary research on K150del variant of activated protein C

doi: 10.18632/aging.202904

Figure Lengend Snippet: Anti-inflammatory activity of APC derivatives using cellular assays. The anti-inflammatory activity of different concentrations of APC derivatives was assessed using four cellular assays as described in the “Methods” section. All results are presented as means ± SD of three different experiments. The significance level, * P < 0.05, ** P < 0.01 and *** P < 0.001, represents differences in the APC-DEL group compared to the APC-WT group. ( A ) Protective effects of APC-WT and APC-DEL on the endothelial cell permeability in response to LPS. The permeability was determined spectrophotometrically at 650 nm. ( B ) Effects of APC-WT and APC-DEL on LPS-induced expression of cell adhesion molecules, ICAM-1, VCAM-1, and E-selectin, on the surface of endothelial cells. The expression was measured using an ELISA kit and detected at 450 nm. ( C ) The inhibitory effect of APC-WT and APC-DEL on the adherence of THP-1 cells to LPS-activated endothelial cells. The percentage of adherent THP-1 cells was calculated using the following formula: % adherence = (adherent signal/total signal) x 100. ( D ) The inhibitory effect of APC-WT and APC-DEL on LPS-induced NF-κB activation. The degree of activation of NF-κB was measured using an ELISA kit and detected at 450 nm.

Article Snippet: Mouse anti-human E-selectin monoclonal antibody and staurosporine were bought from Millipore (MO, USA).

Techniques: Activity Assay, Permeability, Expressing, Enzyme-linked Immunosorbent Assay, Activation Assay