ABSTRACT
This protocol describes how to prepare mouse brain tissue for quantification of multiple inflammatory mediators using a multiplex bead-based immunoassay. It is important to have methods that allow quantification of multiple analytes from small amounts of tissue. Bio-Plex is a Luminex xMAP-based multiplex bead-based immunoassay technology that permits simultaneous analysis of up to 100 analytes from a single tissue sample. This assay has been used extensively to investigate analytes in plasma and serum samples as well as cultured and primary cells. Here, we describe a method for simultaneous analysis of 33 different inflammatory cytokines and chemokines from mouse brain tissue using the Bio-Plex Pro Mouse Chemokine Panel 33-Plex.
Subject(s)
Biological Assay/methods , Chemokines/analysis , Cytokines/analysis , High-Throughput Screening Assays/methods , Malaria, Cerebral/diagnosis , Animals , Biological Assay/instrumentation , Biomarkers/analysis , Brain/immunology , Brain/pathology , Chemokines/immunology , Cytokines/immunology , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay/instrumentation , Enzyme-Linked Immunosorbent Assay/methods , High-Throughput Screening Assays/instrumentation , Humans , Malaria, Cerebral/immunology , Malaria, Cerebral/parasitology , Malaria, Cerebral/pathology , Mice , Microspheres , Plasmodium berghei/immunologyABSTRACT
The Bio-Plex(®) system utilizes xMAP technology to permit the multiplexing of up to 100 different analytes. Multiplex analysis gives researchers the ability to look at analytes simultaneously providing more information from less sample volume in less time than traditional immunoassay methods. Similar to ELISA, xMAP utilizes an antibody sandwich for detection but differs from ELISA in capture substrate and detection method. Rather than a flat surface, Bio-Plex(®) assays make use of differentially detectable bead sets as a substrate capturing analytes in solution and employs fluorescent methods for detection. These bead sets identify the analytes and detection antibodies are used to measure the quantity of analyte. The use of differentially detectable beads enables the simultaneous identification and quantification of many analytes in the same sample.
