Phone:
+1 877 302 8632
Fax:
+1 888 205 9894 (Toll-free)
E-Mail:
orders@antibodies-online.com

CytoSelect™ 96-well Leukocyte-endothelium Adhesion Kit

BCA Fluorometric Cell Samples, Serum Quantitative
Catalog No. ABIN3198656
  • Detection Method
    Fluorometric
    Application
    Biochemical Assay (BCA)
    Brand
    CytoSelect™
    Sample Type
    Cell Samples, Serum
    Analytical Method
    Quantitative
    Characteristics
    CytoSelect™ Leukocyte-endothelium Adhesion Assay provides a robust system for the quantitative determination of leukocyte-endothelium interactions. The kit contains sufficient reagents for the evaluation of 100 assays in a 96-well plate.
    Components
    1. 500X LeukoTracker™ Solution : One 100 μL tube
    2. Gelatin Solution : One 12 mL bottle of sterile 0.1 % Gelatin in 1X PBS
    3. 4X Lysis Buffer : One 10 mL bottle 3
    4. 10X Wash Buffer : One 20 mL bottle
    5. TNFα : One 100 μL tube of 10 μg/mL TNFα in sterile 1X PBS/0.1%BSA
    Material not included
    1. Endothelial cells and cell culture medium
    2. 96-well or 48-well plate
    3. Serum free medium, such as DMEM containing 0.5 % BSA, 2 mM CaCl2 and 2 mM MgCl2
    4. Sterile 1X PBS
    5. Cell culture incubator (37 °C, 5 % CO2 atmosphere)
    6. Light microscope
    7. 96-well plate suitable for a fluorescence plate reader
    8. Fluorescence plate reader
  • Application Notes
    Optimal working dilution should be determined by the investigator.
    Comment

    • Quantify interactions between leukocytes and the endothelium
    • Fully quantitative with no manual cell counting
    • Highly sensitive results on a fluorescence plate reader

    Reagent Preparation

    1X Wash Buffer: Prepare a 1X Wash Buffer by diluting the provided 10X stock 1:10 in deionized water. Store the diluted solution at room temperature. 1X Lysis Buffer: Prepare a 1X Lysis Buffer by diluting the provided 4X stock 1:4 in deionized water. Store the diluted solution at room temperature. Gelatin Coating 1. Under sterile conditions, add 200 μL of the Gelatin Solution to each well of a 48-well tissue culture treated plate, or 100 μL of the Gelatin Solution to each well of a 96-well tissue culture treated plate. 2. Incubate for 60 min at 37 °C in a cell culture incubator. 3. Wash twice with sterile 1X PBS. Aspirate the final wash before use.

    Assay Procedure
    1. Add 50,000-100,000 endothelial cells/well to the Gelatin-coated 48-well or 96-well plate.
    2. Culture cells for 48-72 until the endothelial cells form a monolayer.
    3. Treat endothelial cell monolayer or leukocyte with desired activator or inhibitor for 6-12 hrs. 4 6
    4. Harvest leukocytes and prepare a cell suspension at 1.0 x 10 cells/mL in serum free media. Add LeukoTracker to a final concentration of 1X (for example, add 2 μL of 500X LeukoTracker™ solution to 1.0 mL of leukocyte cell suspension).
    5. Incubate for 60 min at 37 °C in a cell culture incubator. Spin down cells at 1000 rpm for 2 minutes, aspirate the medium and wash cell pellet with serum free media. Repeat the wash twice. 6 Resuspend the cell pellet at 0.25 - 1.0 x 10 cells/mL in serum free media.
    6. Aspirate endothelial culture media and wash once with serum free media. Add 200 μL of the cell suspension to each well already containing the endothelial monolayer.
    7. Incubate for 30-90 min in a cell culture incubator.
    8. Carefully discard or aspirate the media from each well (Note: Do not allow wells to dry). Gently wash each well 3 times with 250 μL 1X Wash Buffer.
    9. (Optional) Count the adherent leukocytes under an inverted fluorescence microscope, average at least three separate fields per well.
    10. Aspirate the final wash and add 150 μL of 1X Lysis Buffer to each well containing cells. Incubate 5 minutes at room temperature with shaking.
    11. Transfer 100 μL of the mixture to a 96-well plate suitable for fluorescence measurement. Read fluorescence with a fluorescence plate reader at 480 nm/520 nm.
    Restrictions
    For Research Use only
  • Storage
    4 °C/-20 °C
    Storage Comment
    LeukoTracker™ Solution and TNFa should be removed from the kit and stored at -20°C immediately. Store all other components at 4°C.
  • Rossignoli, Shang, Gladh, Moessinger, Foroughi Asl, Talukdar, Franzén, Mueller, Björkegren, Folestad, Skogsberg: "Poliovirus Receptor-Related 2: A Cholesterol-Responsive Gene Affecting Atherosclerosis Development by Modulating Leukocyte Migration." in: Arteriosclerosis, thrombosis, and vascular biology, Vol. 37, Issue 3, pp. 534-542, (2017) (PubMed).

    Huang, Qiu, Zeng, Xiao, Shi, Zou, Ye, Liang, Yang, Xu: "Niclosamide inhibits the inflammatory and angiogenic activation of human umbilical vein endothelial cells." in: Inflammation research : official journal of the European Histamine Research Society ... [et al.], Vol. 64, Issue 12, pp. 1023-32, (2016) (PubMed).

    He, Chen, Martin, Zhang, Sangwung, Woo, Tremoulet, Shimizu, Jain, Burns, Shyy: "miR-483 Targeting of CTGF Suppresses Endothelial-to-Mesenchymal Transition: Therapeutic Implications in Kawasaki Disease." in: Circulation research, Vol. 120, Issue 2, pp. 354-365, (2016) (PubMed).

    Cao, Cui, Wu, Zha, Wang, Parks, Yu, Shi, Xue: "Myeloid Deletion of α1AMPK Exacerbates Atherosclerosis in LDL Receptor Knockout (LDLRKO) Mice." in: Diabetes, Vol. 65, Issue 6, pp. 1565-76, (2016) (PubMed).

    Ibrahim, Elshafey, Sellak, Hussein, El-Sherbiny, Abdelsaid, Rizk, Beasley, Tawfik, Smith, Al-Shabrawey: "A lipidomic screen of hyperglycemia-treated HRECs links 12/15-Lipoxygenase to microvascular dysfunction during diabetic retinopathy via NADPH oxidase." in: Journal of lipid research, Vol. 56, Issue 3, pp. 599-611, (2015) (PubMed).

    Campos-Estrada, Liempi, González-Herrera, Lapier, Kemmerling, Pesce, Ferreira, López-Muñoz, Maya: "Simvastatin and Benznidazole-Mediated Prevention of Trypanosoma cruzi-Induced Endothelial Activation: Role of 15-epi-lipoxin A4 in the Action of Simvastatin." in: PLoS neglected tropical diseases, Vol. 9, Issue 5, pp. e0003770, (2015) (PubMed).

    Shah, Romero, Zhu, Duong, Sun, Walsh, Summer: "C1q Deficiency Promotes Pulmonary Vascular Inflammation and Enhances the Susceptibility of the Lung Endothelium to Injury." in: The Journal of biological chemistry, Vol. 290, Issue 49, pp. 29642-51, (2015) (PubMed).

    Sharma-Walia, Chandran, Patel, Veettil, Marginean: "The Kaposi's sarcoma-associated herpesvirus (KSHV)-induced 5-lipoxygenase-leukotriene B4 cascade plays key roles in KSHV latency, monocyte recruitment, and lipogenesis." in: Journal of virology, Vol. 88, Issue 4, pp. 2131-56, (2014) (PubMed).

    Burrows, Maziarz, Hunady, Lehman, Raber, Deans, Vant Hof: "Human multipotent adult progenitor cells transcriptionally regulate fucosyltransferase VII." in: Cytotherapy, Vol. 16, Issue 4, pp. 566-75, (2014) (PubMed).

    Kapitsinou, Sano, Michael, Kobayashi, Davidoff, Bian, Yao, Zhang, Harris, Duffy, Erickson-Miller, Sutton, Haase: "Endothelial HIF-2 mediates protection and recovery from ischemic kidney injury." in: The Journal of clinical investigation, Vol. 124, Issue 6, pp. 2396-409, (2014) (PubMed).

    Cary, Noutai, Salber, Williams, Ngudiankama, Whitnall: "Interactions between endothelial cells and T cells modulate responses to mixed neutron/gamma radiation." in: Radiation research, Vol. 181, Issue 6, pp. 592-604, (2014) (PubMed).

    Wu, Fan, Fang, Wu: "Regulation of microRNA-155 in endothelial inflammation by targeting nuclear factor (NF)-?B P65." in: Journal of cellular biochemistry, Vol. 115, Issue 11, pp. 1928-36, (2014) (PubMed).

    Ghoshal, Rajendran, Odo, Ikuta: "Glycosylation inhibitors efficiently inhibit P-selectin-mediated cell adhesion to endothelial cells." in: PLoS ONE, Vol. 9, Issue 6, pp. e99363, (2014) (PubMed).

    Cao, Wang, Jia, Mondal, Diallo, Hawkins, Das, Parks, Yu, Shi, Shi, Xue: "Inhibiting DNA Methylation by 5-Aza-2'-deoxycytidine ameliorates atherosclerosis through suppressing macrophage inflammation." in: Endocrinology, Vol. 155, Issue 12, pp. 4925-38, (2014) (PubMed).

    Liu, Place, Chen, Brovkovych, Vogel, Muller, Skidgel, Malik, Minshall: "ICAM-1-activated Src and eNOS signaling increase endothelial cell surface PECAM-1 adhesivity and neutrophil transmigration." in: Blood, Vol. 120, Issue 9, pp. 1942-52, (2012) (PubMed).

    Fang, Davies: "Site-specific microRNA-92a regulation of Kruppel-like factors 4 and 2 in atherosusceptible endothelium." in: Arteriosclerosis, thrombosis, and vascular biology, Vol. 32, Issue 4, pp. 979-87, (2012) (PubMed).

    Curatola, Huang, Naftolin: "Dehydroepiandrosterone (DHEA) inhibition of monocyte binding by vascular endothelium is associated with sialylation of neural cell adhesion molecule." in: Reproductive sciences (Thousand Oaks, Calif.), Vol. 19, Issue 1, pp. 86-91, (2012) (PubMed).

    Wang, Malik, Sun, Hu, Reynolds, Minshall, Hu: "Innate immune function of the adherens junction protein p120-catenin in endothelial response to endotoxin." in: Journal of immunology (Baltimore, Md. : 1950), Vol. 186, Issue 5, pp. 3180-7, (2011) (PubMed).

    Xue, Thippegowda, Hu, Bachmaier, Christman, Malik, Tiruppathi: "NF-kappaB regulates thrombin-induced ICAM-1 gene expression in cooperation with NFAT by binding to the intronic NF-kappaB site in the ICAM-1 gene." in: Physiological genomics, Vol. 38, Issue 1, pp. 42-53, (2009) (PubMed).

  • Background
    Leukocyte extravasation into perivascular tissue plays a key role in inflammatory diseases. This recruitment requires leukocyte interaction with vascular endothelium and consists of multiple, consecutive processes including the capture of circulating leukocytes, subsequent leukocyte rolling, arrest, firm adhesion and transmigration (Figure 1). This multistep paradigm is realized by sequential activation-dependent interactions between endothelial cell adhesion molecules and their specific ligands on leukocytes. The first step of transient adhesion and rolling is known to be mediated by an interaction of leukocyte or endothelial cell selectins and their oligosaccharide-bearing ligands. Arrest and firm adhesion of leukocytes to endothelium is dependent on the activation of β2 integrins like Mac-1 or LFA-1 on the leukocyte cell surface, followed by interaction with endothelial cell proteins belonging to the Ig superfamily such as ICAM-1.
You are here:
Support