Cytokine Measurement Using Cytometric Bead Arrays
Cytokines can be measured by enzyme-linked immunosorbent assay (ELISA) or multiplex assay. Both techniques are commonly used in immunology to detect the presence of antibody or antigen in a sample. However, multiplex bead array technology provides the mea
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1. Introduction To measure cytokines secreted from cells, researchers commonly use conventional ELISA (enzyme-linked immunosorbent assay) techniques. ELISA methods are restricted to measuring one cytokine at a time and, moreover, samples are rapidly used up when several ELISA reactions have to be performed. Although new plates are available to measure multiple cytokines in human samples in a single ELISA, it is limited to measuring eight cytokines per well (1). The launch of a multiplex cytometric bead array (CBA) system by BD Biosciences and the Luminex system by the Luminex Corporation have provided the possibility of analysing several proteins in a small volume of fluid (2–5). CBA assays provide the means to measure up to 30 proteins simultaneously in small volume samples (typically 25–50 μL) and it is possible to quantify a variety of soluble and intracellular proteins, such as cytokines, chemokines, growth factors, and phosphorylated proteins (6, 7).
Alexandra C. Brand and Donna M. MacCallum (eds.), Host-Fungus Interactions: Methods and Protocols, Methods in Molecular Biology, vol. 845, DOI 10.1007/978-1-61779-539-8_29, © Springer Science+Business Media, LLC 2012
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L. Castillo and D.M. MacCallum
Moreover, CBA sets are available to detect cytokines for several species, such as human, rat, and mouse. CBA assays were validated for measurement of cytokines from serum and tissue culture; however, this method is also useful for measurement of cytokines in supernatants from organ homogenates (8). Taking advantage of the CBA assay, we have measured cytokines in biological samples in mice infected with Candida albicans (8). C. albicans is the most common aetiological agent of candidiasis. In the intravenous (IV) challenge model for experimental C. albicans infection in mice, the fungus typically proliferates in kidneys but is cleared from the spleen (9, 10), indicating differential host responses in these two organs. However, few studies have associated tissue damage with tissue-specific immune response (8, 11). We have studied production of cytokines and chemokines in kidneys, spleen, and serum from BALB/c mice infected intravenously with different C. albicans strains (8). The use of the CBA assay allowed us to quantify a range of cytokines and chemokines in supernatants from homogenised kidneys and spleens, or in serum, at intervals up to 72 h post-challenge. Here we present the protocol to carry out the CBA assay using such biological samples.
2. Materials 2.1. Reagents
1. CBA Mouse cytokine Flex Sets (BD Biosciences) (see Note 1). 2. BD CBA Mouse/Rat Soluble Protein Master Buffer kit (see Note 2). 3. 1.8-mL microcentrifuge tubes. 4. 30–50 mL sterile containers (e.g. Universal tubes or Falcon tubes). 5. 96-well round-bottomed plates. 6. Multi-channel pipette (30–300 μL). 7. Blunt-bottomed 1.8-mL tubes containing 500 μL saline plus protease inhibitors (see Note 3). 8. Microcentrifuge tubes containing 0.5 μL protease inhibitor cocktail for collection of blood. Add 0.5 μL protease inhibitor cocktail (Sigm
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