mouse monoclonal against icam‐1 bbig‐ i1(11c81  (R&D Systems)

Journal: Physiological Reports

Article Title: Endothelial cells of pulmonary origin display unique sensitivity to the bacterial endotoxin lipopolysaccharide

doi: 10.14814/phy2.15271

Figure Lengend Snippet: Pulmonary HMVECs display increased ICAM‐1 expression and a stronger and earlier response to LPS than HUVECs. (a) Histogram of ICAM‐1 distribution after 5 h LPS as measured with FACS. Y ‐axis top to bottom represents increasing concentrations of LPS treatment of HUVEC and pulmonary HMVECs (pulmonary) as indicated. Condition 0 represents PBS CTRL treatment. X ‐axis represents the increasing fluorescent intensity of ICAM‐1 measured with anti‐ICAM‐1‐FITC antibody. All cells were selected for PECAM‐1 and VE‐cadherin double positivity. (b) Quantification of ICAM‐1 surface expression after treatment of pulmonary HMVECs (pulmonary) and HUVEC with PBS (CTRL) or 10 ng/mL LPS as measured with FACS. All PBS‐treated HUVEC data points were divided by their average to indicate variation. All pulmonary HMVECs data points were divided by PBS‐treated HUVEC data points for fold difference. The dotted line indicates 1‐fold. ns = non‐significant, ** p < 0.01, *** p < 0.001, n = 4. (c) Number of transmigrated neutrophils during flow TEM assay in HUVEC and HMVEC (Pulmonary) treated as indicated. Each data point represents average neutrophil TEM per field of view per condition. Three fields of view per channel were obtained during time‐lapse imaging, followed by tile scans with 25 additional fields of view per condition and replicate at the endpoint to determine the distribution of neutrophils. Bar represents an average of all experiments per condition combined. nd = not detected, ** p < 0.01, error bars are presented as standard error of mean (SEM), n = 3. (d) Representative images from TEM assay. Black star indicates an area with a small cluster of transmigrated PMNs

Article Snippet: IF: mouse monoclonal against ICAM‐1 clone BBIG‐ I1(11C81) (R&D), chicken α‐mouse‐AF488 (Invitrogen), mouse monoclonal against VE‐cadherin (clone 55‐7H1, BD) conjugated with Alexa647.

Techniques: Expressing, Imaging

Journal: Physiological Reports

Article Title: Endothelial cells of pulmonary origin display unique sensitivity to the bacterial endotoxin lipopolysaccharide

doi: 10.14814/phy2.15271

Figure Lengend Snippet: Pulmonary HMVECs show increased ICAM‐1 expression and efficiently facilitate LPS‐induced neutrophil TEM compared to other types of HMVECs. (a) Quantification of FACS data for ICAM‐1 at treatment with 10 ng/mL LPS. Renal HMVEC data points were divided by their own average to indicate variance. All other data points were divided by renal HMVEC data points to acquire fold difference. All cells had been selected for PECAM‐1 and VE‐cadherin double‐positivity. * p < 0.05, error bars are presented as standard error of mean (SEM), n = 3 except HCEC n = 2. (b) Number of transmigrated neutrophils during flow TEM assay in all organ microvascular ECs as indicated after 5 h treatment with 10 ng/mL LPS. Data points represent PMN‐TEM per field of view (FOV). Three fields of view per channel were obtained during time‐lapse imaging, followed by tile scans with 25 additional fields of view per condition and replicate at the endpoint to determine the distribution of neutrophils. Bars represent average TEM of PMN/FOV for all experiments. * p < 0.05, ** p < 0.01, error bars are presented as standard error of mean (SEM), n = 3–6. (c) Representative images from TEM assay. Black star indicates an area with a small cluster of transmigrated PMNs

Article Snippet: IF: mouse monoclonal against ICAM‐1 clone BBIG‐ I1(11C81) (R&D), chicken α‐mouse‐AF488 (Invitrogen), mouse monoclonal against VE‐cadherin (clone 55‐7H1, BD) conjugated with Alexa647.

Techniques: Expressing, Imaging

Journal: Physiological Reports

Article Title: Endothelial cells of pulmonary origin display unique sensitivity to the bacterial endotoxin lipopolysaccharide

doi: 10.14814/phy2.15271

Figure Lengend Snippet: CDH13 aids neutrophil‐TEM in pulmonary HMVECs (a) Western blot analysis of whole cell lysates from pulmonary HMVECs after 96h from lentiviral transduction with short‐hairpin control (shCtrl) or short‐hairpin CDH13 (shCDH13) and incubated with antibodies against CDH13 and β‐actin. Images were developed on film using ECL. Images were analyzed with ImageJ. X marks the three constructs used for experiments. (b) Quantification of Western blot exampled in Figure . shCtrl and shCDH13 units derived from ImageJ were divided by units for β‐actin to generate arbitrary units. All shCDH13 samples were divided by shCtrl which was set to 1 to indicate knock‐down efficiency. Error bar is presented as standard error of mean (SEM), n = 3 (c) Number of transmigrated neutrophils during flow TEM assay in pulmonary ECs after 5 h treatment with 10 ng/mL LPS. Data points represent PMN‐TEM per field of view (FOV). Three fields of view per channel were obtained during time‐lapse imaging, followed by tile scans with 25 additional fields of view per condition and replicate at the endpoint to determine the distribution of neutrophils. Bars represent average TEM of PMN/FOV for all experiments. *** p < 0.001, error bars are presented as standard error of mean (SEM), n = 3. (d) Representative images from TEM assay. Black star indicates an area with a small cluster of transmigrated PMNs. (e) Western blot analysis and quantification of whole cell lysates from pulmonary HMVECs after 96 h lentiviral transduction with short‐hairpin control (shCtrl) or short‐hairpin CDH13 (shCDH13), PBS or LPS stimulation for 20 h and incubated with antibodies against ICAM‐1 and β‐actin. Images were developed on film using ECL. Images were analyzed with ImageJ. ICAM‐1 units derived from ImageJ were divided by units for β‐actin to generate arbitrary units. AU = arbitrary units, ns = non‐significant, ** p < 0.01, error bars are presented as standard error of mean (SEM), n = 3. (f) Flow cytometry data for ICAM‐1 in pulmonary ECs treated as indicated. PBS‐treated sh‐Ctrl pulmonary HMVECs data points were divided by their own average to indicate variance. All other data points were divided by PBS‐treated sh‐Ctrl pulmonary HMVECs data points to acquire fold difference. All cells had been selected for PECAM‐1 and VE‐cadherin double‐positivity, ns = non‐significant, error bars are presented as standard error of mean (SEM), n = 3

Article Snippet: IF: mouse monoclonal against ICAM‐1 clone BBIG‐ I1(11C81) (R&D), chicken α‐mouse‐AF488 (Invitrogen), mouse monoclonal against VE‐cadherin (clone 55‐7H1, BD) conjugated with Alexa647.

Techniques: Western Blot, Transduction, Incubation, Construct, Derivative Assay, Imaging, Flow Cytometry

Journal: Science Advances

Article Title: Myeloid-derived growth factor inhibits inflammation and alleviates endothelial injury and atherosclerosis in mice

doi: 10.1126/sciadv.abe6903

Figure Lengend Snippet: AKO and DKO mice aged 4 to 6 weeks were fed a WD for 12 weeks (10 mice in each group). ( A and B ) The vasodilatation reaction induced by Ach (A) and SNP (B) ( n = 10). ( C ) Representative images of en face atherosclerotic lesions. ( D ) Quantitative analysis of (C) ( n = 5). ( E ) Representative images of the cross-sectional area of the aortic root ( n = 8). Scale bars, 500 μm. ( F ) Quantitative analysis of (E). ( G ) Representative immunohistochemical staining images of VSMCs [α–smooth muscle actin (α-SMA)], collagen (Masson), macrophages (anti-CD68), and T lymphocytes (anti-CD3) in aortic plaques. Scale bar, 100 μm. ( H ) Quantitative analysis of (G) ( n = 8). ( I and J ) The mRNA levels of adhesion molecules (VCAM-1, ICAM-1, and E-selectin) (I) and inflammation (TNF-α, IL-1β, and IL-6) (J) in MAECs of mice ( n = 5). The data are presented as the means ± SEM. * P < 0.05 and ** P < 0.001.

Article Snippet: The sections from the aortic arch were additionally stained with monoclonal anti–ICAM-1 BBIG-I1 (1:100) or monoclonal anti–VCAM-1 BBIG-V1 (1:200) (R&D Systems) and rat monoclonal anti-CD31 (1:100; ABclonal, ab24590) and mounted in Prolong Gold with DAPI (Life Technologies).

Techniques: Immunohistochemical staining, Staining

Journal: The Journal of Immunology Author Choice

Article Title: Human Endothelial Cell Activation by Escherichia coli and Staphylococcus aureus Is Mediated by TNF and IL-1β Secondarily to Activation of C5 and CD14 in Whole Blood

doi: 10.4049/jimmunol.1502220

Figure Lengend Snippet: E. coli and S. aureus cause dose-dependent EC activation in whole blood. Expression of E-selectin (A and C) and ICAM-1 (B and D) on EC after incubation of E. coli (A and B) or S. aureus (C and D) in growth medium, normal human serum, or in whole blood. Data are given as mean ± range of n = 3 and n = 4 for E. coli and S. aureus, respectively.

Article Snippet: Abs used for flow cytometric detection of EC surface proteins were FITC-conjugated mouse anti-human ICAM-1 (CD54, clone BBIG-I1) and isotype control (FITC-conjugated mouse IgG1, clone 11711), PerCP-conjugated mouse anti-human MCAM (CD146, clone 128018) (from R&D Systems, Minneapolis, MN), PE-conjugated mouse anti-human E-selectin (CD62E, clone 1.2B6) and isotype control (PE-conjugated mouse IgG1, clone 15H6) (both from SouthernBiotech, Birmingham, AL).

Techniques: Activation Assay, Expressing, Incubation

Journal: The Journal of Immunology Author Choice

Article Title: Human Endothelial Cell Activation by Escherichia coli and Staphylococcus aureus Is Mediated by TNF and IL-1β Secondarily to Activation of C5 and CD14 in Whole Blood

doi: 10.4049/jimmunol.1502220

Figure Lengend Snippet: Complement (C5) and TLR (CD14 and TLR4/MD2) inhibition of whole blood attenuates EC activation by E. coli and S. aureus. Monolayers of EC were incubated with whole blood. The C5 inhibitors eculizumab or C5aR1 antagonist, anti-CD14, TLR4/MD2 antagonist eritoran, or combinations of these, as well as control Ab (rituximab) were added to the blood prior to the addition of 105 bacteria/ml E. coli (A and B) or 107 bacteria/ml S. aureus (C and D). E-selectin expression (A and C) and ICAM-1 expression (B and D) data are presented with median values (bars), mean values (+), and error bars from the 10th to 90th percentile with n = 6 donors. *p < 0.05, **p < 0.01, ***p < 0.001 as compared with bacterial incubation without inhibitors.

Article Snippet: Abs used for flow cytometric detection of EC surface proteins were FITC-conjugated mouse anti-human ICAM-1 (CD54, clone BBIG-I1) and isotype control (FITC-conjugated mouse IgG1, clone 11711), PerCP-conjugated mouse anti-human MCAM (CD146, clone 128018) (from R&D Systems, Minneapolis, MN), PE-conjugated mouse anti-human E-selectin (CD62E, clone 1.2B6) and isotype control (PE-conjugated mouse IgG1, clone 15H6) (both from SouthernBiotech, Birmingham, AL).

Techniques: Inhibition, Activation Assay, Incubation, Bacteria, Expressing

Journal: The Journal of Immunology Author Choice

Article Title: Human Endothelial Cell Activation by Escherichia coli and Staphylococcus aureus Is Mediated by TNF and IL-1β Secondarily to Activation of C5 and CD14 in Whole Blood

doi: 10.4049/jimmunol.1502220

Figure Lengend Snippet: Complement (C5)- and TLR (CD14 and TLR4/MD2)-induced EC activation is mediated by TNF and IL-1β. Monolayers of EC were incubated with whole blood and the TNF-blocking Ab infliximab, the IL-1β–blocking Ab canakinumab, a combination of these, or control Ab (rituximab) prior to the addition of 105 bacteria/ml E. coli (A and B) or 107 bacteria/ml S. aureus (C and D). E-selectin expression (A and C) and ICAM-1 expression (B and D) data are presented as in Fig. 2 with n = 6 donors. ***p < 0.001, ****p < 0.0001 as compared with bacterial incubation without inhibitors.

Article Snippet: Abs used for flow cytometric detection of EC surface proteins were FITC-conjugated mouse anti-human ICAM-1 (CD54, clone BBIG-I1) and isotype control (FITC-conjugated mouse IgG1, clone 11711), PerCP-conjugated mouse anti-human MCAM (CD146, clone 128018) (from R&D Systems, Minneapolis, MN), PE-conjugated mouse anti-human E-selectin (CD62E, clone 1.2B6) and isotype control (PE-conjugated mouse IgG1, clone 15H6) (both from SouthernBiotech, Birmingham, AL).

Techniques: Activation Assay, Incubation, Blocking Assay, Bacteria, Expressing

Journal: Nature Communications

Article Title: Endothelial protein kinase MAP4K4 promotes vascular inflammation and atherosclerosis

doi: 10.1038/ncomms9995

Figure Lengend Snippet: ( a , b ) Control and M4K4 KD mice were fed WD for 16 weeks. Aortic arch sections were stained for Cd31 as an endothelial marker (green), Icam-1 (red, ( a )), Vcam-1 (red, ( b )) or DAPI. From left, CD31, Icam-1/Vcam-1, DAPI and merge. Scale bars, 100 μm. Arrowheads indicate Cd31/Icam-1 or Cd31/Vcam-1 colocalization. Images are representative of at least three control and three M4K4 KD animals. DAPI, 4,6-diamidino-2-phenylindole.

Article Snippet: Membranes were immunoblotted with anti-ICAM-1 BBIG-I1 (1:1,000), VCAM-1 BBIG-V1 (1:1,000), E-selectin BBIG-E4 (1:1,000) (R&D systems), MAP4K4 A301-503A (1:2,000) (Bethyl), VE-cadherin 2500 (1:2,000), p-JNK1/2 4668 (1:1,000), total JNK1/2 9258 (1:1,000), p-p38 MAPK 4631 (1:1,000), p-IκBα 9246 (1:1,000), IκBα 4814 (1:1,000), p-p65 3039 (1:1,000), p65 8242 (1:1,000), HGK 3485 (1:1,000), p-Erk 4370 (1:1,000) (Cell Signaling Technology), total p38 MAPK sc-535 (1:1,000), total Erk1 sc-93 (1:4,000) (Santa Cruz) or lamin-β1 ab16048 (1:1,000) (Abcam) antibodies.

Techniques: Staining, Marker

Journal: Nature Communications

Article Title: Endothelial protein kinase MAP4K4 promotes vascular inflammation and atherosclerosis

doi: 10.1038/ncomms9995

Figure Lengend Snippet: HUVECs were treated with scrambled or MAP4K4 siRNA, and cells were stimulated with 1 or 10 ng ml −1 TNF-α for the indicated times. ( a ) Cells were seeded onto transwell chambers. Confluent cells were treated overnight with 10 ng ml −1 TNF-α or left untreated, and FITC-labelled dextran that migrated through the HUVEC monolayer was measured. The data represent the mean fluorescence intensity±s.e.m. (analysis of variance (ANOVA) * P <0.05, N =4). ( b ) THP-1 monocytes were stained with calcein green and adhered to activated endothelium for 30 min. Fluorescence microscopy was performed to determine the number of adherent THP-1 monocytes per microscopic field (× 100). The data represent the mean±s.e.m. as normalized to the unstimulated control time point (ANOVA ** P <0.01, **** P <0.0001, N =5). ( c – f ) Messenger RNA was extracted and quantitative RT–PCR was performed for ( c ) MAP4K4 , ( d ) ICAM-1 , ( e ) VCAM-1 and ( f ) SELE . The data represent the mean±s.e.m. as normalized to RPLP0 (* P <0.05, ** P <0.005, # P <0.0001, N =5–7). ( g ) Immunoblots were performed for ICAM-1, VCAM-1 and E-selectin. ( h ) Densitometric analyses from g . The data represent the means±s.e.m. as normalized to VE-cadherin (* P <0.05, N =4–6). ( i ) Biochemical fractionations were performed, and nuclear fractions were immunoblotted for MAP4K4, p-p65, total p65, p50 and lamin-β1. ( j ) Densitometric analyses represent the mean±s.e.m. for the 60-min time point as normalized to lamin-β1 (* P <0.05, ** P <0.005, N =3). ( k ) NFκB-luciferase and SV40-Renilla were transfected into HUVECs after treatment with scrambled or MAP4K4 siRNA. Cells were left unstimulated or stimulated with 10 ng ml −1 TNF-α overnight before luciferase and Renilla measurement. The data represent the mean±s.e.m. from four independent experiments. ( l – n ) HUVECs were transfected with MAP4K4 siRNA and stimulated or not with 1 ng ml −1 TNF-α for 1 h. IgG, p65 and histone antibodies were used to immunoprecipitate chromatin and RT–PCR was used to amplify ( l ) E-selectin, ( m ) VCAM-1 and ( n ) IκBα promoters. The data represent the mean±s.e.m. as normalized to input (* P <0.05, N =3–4).

Article Snippet: Membranes were immunoblotted with anti-ICAM-1 BBIG-I1 (1:1,000), VCAM-1 BBIG-V1 (1:1,000), E-selectin BBIG-E4 (1:1,000) (R&D systems), MAP4K4 A301-503A (1:2,000) (Bethyl), VE-cadherin 2500 (1:2,000), p-JNK1/2 4668 (1:1,000), total JNK1/2 9258 (1:1,000), p-p38 MAPK 4631 (1:1,000), p-IκBα 9246 (1:1,000), IκBα 4814 (1:1,000), p-p65 3039 (1:1,000), p65 8242 (1:1,000), HGK 3485 (1:1,000), p-Erk 4370 (1:1,000) (Cell Signaling Technology), total p38 MAPK sc-535 (1:1,000), total Erk1 sc-93 (1:4,000) (Santa Cruz) or lamin-β1 ab16048 (1:1,000) (Abcam) antibodies.

Techniques: Fluorescence, Staining, Microscopy, Quantitative RT-PCR, Western Blot, Luciferase, Transfection, Reverse Transcription Polymerase Chain Reaction

Journal: Nature Communications

Article Title: Endothelial protein kinase MAP4K4 promotes vascular inflammation and atherosclerosis

doi: 10.1038/ncomms9995

Figure Lengend Snippet: ( a , b ) Control and M4K4 KD mice were fed WD for 16 weeks. Aortic arch sections were stained for Cd31 as an endothelial marker (green), Icam-1 (red, ( a )), Vcam-1 (red, ( b )) or DAPI. From left, CD31, Icam-1/Vcam-1, DAPI and merge. Scale bars, 100 μm. Arrowheads indicate Cd31/Icam-1 or Cd31/Vcam-1 colocalization. Images are representative of at least three control and three M4K4 KD animals. DAPI, 4,6-diamidino-2-phenylindole.

Article Snippet: Aortic roots were additionally stained with haematoxylin and eosin or trichrome by the UMASS morphology core, with rat anti-mouse CD68 (1:200) (AbD Serotec, clone FA-11) and Cy3-smooth muscle actin (Sigma S6198) (1:200), or with anti-ICAM-1 BBIG-I1 (1:200) or VCAM-1 BBIG-V1 (1:200) (R&D systems) and rat anti-mouse CD31 (1:400) (BD) and mounted in Prolong Gold with DAPI (Life Technologies).

Techniques: Staining, Marker

Journal: Nature Communications

Article Title: Endothelial protein kinase MAP4K4 promotes vascular inflammation and atherosclerosis

doi: 10.1038/ncomms9995

Figure Lengend Snippet: HUVECs were treated with scrambled or MAP4K4 siRNA, and cells were stimulated with 1 or 10 ng ml −1 TNF-α for the indicated times. ( a ) Cells were seeded onto transwell chambers. Confluent cells were treated overnight with 10 ng ml −1 TNF-α or left untreated, and FITC-labelled dextran that migrated through the HUVEC monolayer was measured. The data represent the mean fluorescence intensity±s.e.m. (analysis of variance (ANOVA) * P <0.05, N =4). ( b ) THP-1 monocytes were stained with calcein green and adhered to activated endothelium for 30 min. Fluorescence microscopy was performed to determine the number of adherent THP-1 monocytes per microscopic field (× 100). The data represent the mean±s.e.m. as normalized to the unstimulated control time point (ANOVA ** P <0.01, **** P <0.0001, N =5). ( c – f ) Messenger RNA was extracted and quantitative RT–PCR was performed for ( c ) MAP4K4 , ( d ) ICAM-1 , ( e ) VCAM-1 and ( f ) SELE . The data represent the mean±s.e.m. as normalized to RPLP0 (* P <0.05, ** P <0.005, # P <0.0001, N =5–7). ( g ) Immunoblots were performed for ICAM-1, VCAM-1 and E-selectin. ( h ) Densitometric analyses from g . The data represent the means±s.e.m. as normalized to VE-cadherin (* P <0.05, N =4–6). ( i ) Biochemical fractionations were performed, and nuclear fractions were immunoblotted for MAP4K4, p-p65, total p65, p50 and lamin-β1. ( j ) Densitometric analyses represent the mean±s.e.m. for the 60-min time point as normalized to lamin-β1 (* P <0.05, ** P <0.005, N =3). ( k ) NFκB-luciferase and SV40-Renilla were transfected into HUVECs after treatment with scrambled or MAP4K4 siRNA. Cells were left unstimulated or stimulated with 10 ng ml −1 TNF-α overnight before luciferase and Renilla measurement. The data represent the mean±s.e.m. from four independent experiments. ( l – n ) HUVECs were transfected with MAP4K4 siRNA and stimulated or not with 1 ng ml −1 TNF-α for 1 h. IgG, p65 and histone antibodies were used to immunoprecipitate chromatin and RT–PCR was used to amplify ( l ) E-selectin, ( m ) VCAM-1 and ( n ) IκBα promoters. The data represent the mean±s.e.m. as normalized to input (* P <0.05, N =3–4).

Article Snippet: Aortic roots were additionally stained with haematoxylin and eosin or trichrome by the UMASS morphology core, with rat anti-mouse CD68 (1:200) (AbD Serotec, clone FA-11) and Cy3-smooth muscle actin (Sigma S6198) (1:200), or with anti-ICAM-1 BBIG-I1 (1:200) or VCAM-1 BBIG-V1 (1:200) (R&D systems) and rat anti-mouse CD31 (1:400) (BD) and mounted in Prolong Gold with DAPI (Life Technologies).

Techniques: Fluorescence, Staining, Microscopy, Quantitative RT-PCR, Western Blot, Luciferase, Transfection, Reverse Transcription Polymerase Chain Reaction

Journal: Nutrition & Metabolism

Article Title: A novel anti-inflammatory natural product from Sphaeranthus indicus inhibits expression of VCAM1 and ICAM1, and slows atherosclerosis progression independent of lipid changes

doi: 10.1186/s12986-015-0018-1

Figure Lengend Snippet: Sphaeranthus Indicus extract inhibits TNF-α-induced production of adhesion molecules human umbilical vein endothelial cells. Confluent HUVECs in 96-well fibronectin–coated plates were pretreated with various concentrations of S. indicus or 7-HF for 30 min before being stimulated with 1 ng/mL TNF-α for the indicated time. The expressions of ICAM-1 and E-selectin were evaluated after TNF-α stimulation for 4 h, and expression of VCAM-1 was evaluated after 6 h of stimulation. The 50 % inhibitory concentration (IC 50 ) values were calculated by a nonlinear regression method using GraphPad software

Article Snippet: Anti-ICAM-1 (clone BBIG-I1) anti-VCAM-1 (clone BBIG-V1) and anti-E-selectin (clone BBIG-E4), isotype control mouse IgG 1 (clone 11711.11), the secondary antibody (anti-mouse IgG-HRP antibody) and bacteria derived recombinant human TNF-α were products of R&D Systems (Minneapolis, MN).

Techniques: Expressing, Concentration Assay, Software