To investigate leukocyte adhesion, we used a human flow chamber system. In brief, glass capillaries were coated with P-selectin (20 µg/ml; R&D Systems) and VCAM-1 (20 µg/ml; R&D Systems) or P-selectin (20 µg/ml; R&D Systems), VCAM-1 (20 µg/ml; R&D Systems) and SDF1 (20 µg/ml; R&D Systems) for 2 h. Chambers were blocked with 1% casein (Thermo Fisher Scientific) for 1 h and afterward perfused with isolated CD4 cells at a constant shear stress of 1 dyne/cm2. To investigate the capturing of CD4 cells, flow chambers were coated with P-selectin (20 µg/ml) alone and were perfused with isolated CD4 cells at a constant shear stress of 1 dyne/cm2 for 2 min and, subsequently, the number of rolling cells per field of view was determined. Videos were obtained with an inverted TS100 transmission light microscope (Nikon) equipped with a 10×/0.25 NA objective and a digital camera (Pixelfly; Cooke Corporation).
Vcam 1
Manufactured by R&D Systems
Sourced in United States, United Kingdom
VCAM-1 is a cell surface glycoprotein that plays a role in cell-cell adhesion. It is expressed on the surface of endothelial cells and functions as a receptor for integrins.
Automatically generated - may contain errors
Lab products found in correlation
Icam 1, by R&D Systems (23 mentions)
E selectin, by R&D Systems (5 mentions)
Mcp 1, by R&D Systems (4 mentions)
P selectin, by R&D Systems (3 mentions)
Interleukin 6 (il 6), by R&D Systems (3 mentions)
Human α thrombin, by Enzyme Research (3 mentions)
Syndecan 1, by Diaclone (2 mentions)
Icam 1, by Santa Cruz Biotechnology (2 mentions)
Timp 1, by R&D Systems (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Il 1β, by R&D Systems (2 mentions)
Vascular endothelial growth factor (vegf), by R&D Systems (2 mentions)
Mmp 9, by R&D Systems (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Jurkat, by American Type Culture Collection (1 mentions)
Pecam 1, by R&D Systems (1 mentions)
Human serum albumin (hsa), by Merck Group (1 mentions)
Tcs sp5 2 laser scanning microscope, by Leica camera (1 mentions)
55 protocols using vcam 1
1
Leukocyte Adhesion Dynamics Under Flow
2
Synthesis and Characterization of Integrin Activator 7HP349
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The complete chemical synthesis of integrin activator 7HP349 is included in the patent50 . For all in vitro assays, compounds were dissolved in DMSO to make a series of stock solutions such that a 1:100 dilution in assay buffer or media would yield the desired final working concentrations in 1% DMSO (vehicle). For in vivo studies, 7HP349 was formulated in a vehicle made from individual components on a weight/weight basis. The vehicle is a PBS solution, pH 7.4, containing 17% Tween 80, 16% glycerol. 7HP349 was prepared at 0.02, 0.2, and 2 mg/g (Ovalbumin study) or 0.5 mg/g (Chagas study). Vehicle and 7HP349 solutions were filtered through a 0.22 µm filter and pipetted into sterile septum vials. The final compound concentration (mg/mL) was verified by HPLC compared to a standard curve. Human VCAM-1 and ICAM-1 were purchased from R&D Systems (Minneapolis, MN). Antibodies were purchased from ABD Serotec (Raleigh, NC) (HP2/1 [anti-α4] and 38 [anti-αL]). The mAb 33B6 [anti-β1] was a gift from B. McIntyre (MD Anderson Cancer Center, Houston, TX). The cell lines Jurkat, HSB, and 70Z/3 were obtained from American Type Culture Collection (Manassas, VA) and were maintained in recommended culture media.
3
Glycocalyx and Endothelial Dysfunction Markers
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Using enzyme-linked immunosorbent assays, we measured serum hyaluronan (R&D Systems, Minneapolis, MN) and syndecan-1 (Diaclone, Besancon, France) as markers of the glycocalyx, and serum von Willebrand Factor (vWF), (Assaypro, Cambridge, MA) and vascular cell adhesion molecule (VCAM-1), ( R&D Systems, Minneapolis, MN) as markers of endothelial dysfunction. All measures were performed in duplicate.
4
Quantifying Cell Adhesion Molecules
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5
CLL Cell Adhesion Assay with VCAM-1
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Falcon culture slides were coated with 7.5 µg/ml VCAM-1 (R&D Systems) overnight, washed, and blocked with 2% human serum albumin (Merck Millipore). Purified CLL cells were incubated for 1 h with or without 1 µM ibrutinib, added to the slides, and allowed to adhere for 30 min at 37°C in the presence or not of 5 µg/ml F(ab)2 anti-IgM before fixation with 4% paraformaldehyde. Slides were stained with primary anti-CD49d antibody (clone AHP1225; Bio-Rad) and Cy3-conjugated secondary anti–rabbit antibody. Images were taken with an Olympus IX81 microscope. For quantification, high-resolution images for cluster analysis were acquired on a Leica TCS SP5 II laser scanning microscope using a 63×/1.4-NA oil-immersion objective (Leica), and the number of clusters was analyzed using ImageJ software.
6
Microfluidic Adhesion Assay for CLL Cells
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Six-channel μ-slides (Ibidi) were coated with VCAM-1 (R&D Systems) at 4°C overnight, washed, and blocked with 2% human serum albumin. Purified CLL cells were incubated with or without 1 µM ibrutinib and 5 µg/ml F(ab)2 anti-IgM. The cells were perfused at 0.5 dyn/cm2 for 1 min at 37°C over the coated substrate. The perfusion period was recorded and digitalized. Analysis of the video-recorded segments was done using customized image analysis software (Wimasis). Arresting cells were defined as cells remaining stationary for at least 3 s on the substrate as previously described (Hartmann et al., 2009 (link); Ganghammer et al., 2015 (link)).
7
Molecular Markers of Angiogenesis and Inflammation
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HEGU [7 (link)] and licoricidin [10 (link)] were prepared as previously described. Antibodies against VEGF-A, platelet endothelial cell adhesion molecule (PECAM-1, CD31), MMP-9, TIMP-1, ICAM-1, and VCAM-1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against Ki67 and HIF-1α were obtained from Abcam (Cambridge, UK). An antibody against mouse CD45 and enzyme-linked immunosorbent assay (ELISA) kits for mouse/rat MMP-9, TIMP-1, ICAM-1, and VCAM-1 were purchased from R&D Systems (Minneapolis, MN, USA). Antibodies to iNOS and COX-2 were obtained from BD Transduction Laboratories (Lexington, KY, USA). If not otherwise specified, all other materials were purchased from Sigma (St. Louis, MO, USA).
8
Biomarker Panel for Chronic Disease
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Serum samples of creatinine, albumin (bromocresol purple), calcium, phosphate, intact parathyroid hormone (iPTH), ferritin, cholesterol, triglyceride (TG), hemoglobin, high-sensitivity CRP (hsCRP; by nephelometry assay; CV, 5%) were measured by routine methods at the Department of Laboratory Medicine, Karolinska University Hospital at Huddinge. Commercial ELISA kits were used to determine serum vascular cell adhesion protein-1 (VCAM-1) (R&D Systems, Minneapolis, MN). Plasma concentrations of IL-6 (CV, 4%), TNF, (CV, 2%–5 %) and insulin-like growth factor-1 (IGF-1, CV, 4.3%) were measured on an Immulite TM Automatic Analyzer (Siemens Healthcare; Diagnostics Products Ltd.) according to the manufacturer’s instructions.
9
Vascular Biomarkers in Metabolic Profiling
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TC, HDL-C, TG, and Fasting plasma glucose concentration (FPG) were measured by the autoanalyser (HITACHI, Tokyo, Japan) with commercially available kits (Randox Laboratories, Crumlin, UK). Serum sPLA2-IIa protein was determined using a sandwich-type enzyme-linked immunosorbent assay (ELISA) kit (Cayman Chemicals, Ann Arbor, MI, USA). Serum sPLA2 activity was measured by a selective fluorometric assay (Cayman Chemicals) by using diheptanoyl thio-phosphocholine as fluorescent substrate. We used a solution of 0.1U bee venom PLA2 as a positive control to hydrolyze fluorescent substrate completely. The hydrolysis of substrate in the absence of serum sample was used as negative control. The intra-assay coefficient of variation was 4.2%. ELISAs were used to determine VCAM-1, ICAM-1, E-selectin, and P-selectin levels (R & D Systems, Minneapolis, MNUSA). All measurements were performed according to the manufacturers’ instructions. Each sample was tested in duplicate, and the means of the duplicates were used for the statistical analysis.
10
Evaluating Immune Cell Activation and Apoptosis
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The reagents were as follows: fibronectin (FN) (Sigma; Rehovot, Israel) VCAM-1(R&D Biosystems, Minneapolis, MN, USA); BSA (Sigma); XTT cell proliferation kit (Biological Industries, Bet Haemek, Israel); FAM FLICA Caspase-3,7 and caspase-1 detection kit (Immunochemistry Technologies LLC, MN, USA); anti Cd49d, anti cd49e (Serotec, NC, USA); Staphylococcus aureus Cowan I (SAC) (Calbiochem-Behring Corp., La Jolla, CA, USA). LPS (Sigma); mouse anti-rat CD68 (ED1) (Serotec); rabbit anti-rat FN (Cedarlane, Burlington, ON, Canada); anti-mouse IL-18 (R&D Biosystems); anti-mouse IL-1β (Santa Cruz, Santa Cruz, CA, USA); anti αTubulin (Santa Cruz); anti-rat Thy-1.1(Cederlane); sheep anti-rat GBM (Probetex, San Antonio, TX, USA); rat IL-1β, IL-18, and TNFα ELISA kits (BiosourceThermo Fisher, Waltham, MA, USA); rat connective tissue growth factor (CTGF) and monocyte chemoattractant protein-1 (MCP-1) ELISA kits (MyBiosource, San Diego CA, USA). Creatinine and albumin assay kits (Abcam, San Francisco, CA, USA); AS101 (supplied by M. Albeck, Bar-Ilan University, Ramat Gan, Israel).
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