Recombinant human ICAM-1-Fc, E-selectin-Fc, and CXCL8/IL-8 were purchased from R&D Systems (Minneapolis, MN; Catalog No. 720-IC, 724-ES, and 208-IL respectively). Protein A/G was purchased from Fischer Scientific (Pittsburgh, PA). BS3 crosslinker, Alexa Fluor 488 Phalloidin and Vybrant DiI Cell-Labeling solution was purchased from life technologies (Grand Island, NY). Antibodies used in flow cytometry, FITC mouse anti-human CD106 (VCAM-1), PE-Cy5 mouse anti-human CD62E (E-selectin), and PE-Cy5 mouse anti-human CD54 (ICAM-1) were purchased from BD Biosciences (San Jose, CA) while Alexa Fluor 488 mouse anti-human CD11a/CD18 (mAb24, LFA-1), PE mouse anti-human CD162 (PSGL-1), and PE-Cy5 mouse anti-human CD62L (L-selectin) were purchased from Biolegend (San Diego, CA). Antibodies were used at a saturating concentration of 5 μg/ml or per manufacturer’s instruction. Human atrial natriuretic peptide (ANP) was purchased from BACHEM (Torrance, CA). Normal human primary umbilical vein endothelial cells (HUVEC) were purchased from ATCC (Manassas, VA). Recombinant human IL-1β was purchased from eBioscience (San Diego, CA).
Recombinant human icam 1 fc
Manufactured by R&D Systems
Sourced in United States
Recombinant human ICAM-1-Fc is a fusion protein consisting of the extracellular domain of human intercellular adhesion molecule-1 (ICAM-1) and the Fc region of human IgG1. It is produced in a mammalian cell expression system.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Cxcl8 il 8, by R&D Systems (1 mentions)
Protein a g, by Thermo Fisher Scientific (1 mentions)
Vybrant dii cell labeling solution, by Thermo Fisher Scientific (1 mentions)
Lsm 510 confocal microscope, by Zeiss (1 mentions)
Anti phosphotyrosine 4g10, by Merck Group (1 mentions)
Anti crkii, by Santa Cruz Biotechnology (1 mentions)
Anti rap1, by Merck Group (1 mentions)
Anti hemagglutinin ha, by Abcam (1 mentions)
Anti gfp, by Thermo Fisher Scientific (1 mentions)
Anti cd3, by BioLegend (1 mentions)
Anti α tubulin b 7, by Santa Cruz Biotechnology (1 mentions)
Tnf α, by R&D Systems (1 mentions)
Sb203580, by Cell Signaling Technology (1 mentions)
5 protocols using recombinant human icam 1 fc
1
Endothelial Cell Adhesion Molecule Assay
2
T cell adhesion and imaging protocol
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Chambered coverslips (Labtek, Nunc–Roskilde, Denmark) were coated overnight at 4 °C with recombinant human ICAM-1-Fc (R&D Systems–Minneapolis, MN, USA) and anti-CD3 (clone OKT3, Mabtech) diluted at 3 μg ml−1 and 1 μg ml−1 in PBS, respectively, and blocked with PBS 3% BSA. T cells were allowed to settle for 25 min at 37 °C and non-attached T cells were removed by gentle washing. Cells were analysed by IRM using a Zeiss LSM 510 confocal microscope with a × 63 NA1.4 Plan-Apochromat oil immersion objective. IRM images were obtained using the 633-nm laser line in conjunction with a FT 561 dichroic and an open emission filter. In other experiments, coverslips were coated with only human ICAM-1-Fc and the T cells were treated with 0.5 mM of Manganese (II) chloride tetrahydrate (Mn2+, Sigma Aldrich-Diegem, Belgium) in the following buffer: 20 mM Hepes, 140 mM NaCl and 2 mg ml−1 of glucose.
3
Antibody Characterization for T Cell Signaling
4
Multivalent Polymer-Mediated Cell Adhesion
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The multivalent polymers PAA [HOCH2(HOCH)4CH2NH-PAA] and Ley-PAA, and the multivalent biotinylated polymers PAA-biotin and Ley-PAA-biotin were purchased from GlycoTech Corporation (Gaithersburg, MD, USA). Recombinant human ICAM-1-Fc and TNF-α were purchased from R&D Systems (Minneapolis, MN, USA). The p38 MAPK inhibitor SB203580 and anti-phosphor-p38 MAPK (Thr180/Tyr182) (D3F9) mAb were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-p38 MAPK polyclonal Ab (C-20), anti-TM (D-3), anti-L-selectin (lam1-116) and anti-α-tubulin (B-7) mAbs were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-TS1/18 mAb and Alexa Fluor 488- and 546-conjugated secondary antibodies were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Anti-MOMA-2 mAb was purchased from Abcam (Cambridge, UK).
5
T Cell Cytokine Production Assay
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Maxisorp flat-bottom 96-well plates were coated overnight at 4 °C with 50 μl of anti-CD3 mAb (Clone OKT3, Mabtech) diluted at 0.5 μg ml−1 in PBS with increasing doses of recombinant human ICAM-1-Fc (0.3–15 μg ml−1; R&D Systems). T cells were cultured at 105 cells per well for 3 h to test their ability to produce or secrete cytokines.
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