Optimization of multiplexed bead-based cytokine immunoassays for rat serum and brain tissue

Abstract

The ability to simultaneously quantify multiple signaling molecule protein levels from microscopic neural tissue samples would be of great benefit to deciphering how they affect brain function. This follows from evidence that indicates signaling molecules can be pleiotropic and can have complex interactive behavior that is regionally and cellularly heterogeneous. Multiplexed examination of tissue proteins has been exceedingly difficult because of the absence of available techniques. This void now has been removed by the commercial availability of bead-based immunoassays for targeted proteins that allow analyses of up to 100 (6-150 kDa) proteins from as little as 12 μl. Thus far used only for sera (human and mouse) and culture media, we demonstrate here that sensitive (as low as 2 pg/ml), wide-ranging (up to 2-32 000 pg/ml), accurate (8% intra-assay covariance) and reliable (4-7% inter-assay covariance) measurements can be made of nine exemplary cytokines (e.g., IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, GM-CSF, IFN-γ, TNF-α) simultaneously not only from rat serum but, for the first time, also brain tissue. Furthermore, we describe animal handling procedures that minimize stress as determined by serum glucocorticoid levels since they can influence cytokine expression.

Multiplexed Cytokine Protein Expression Profiles from Spreading Depression in Hippocampal Organotypic Cultures

“…Our intent with their use was to begin determining whether brain tissue and SD could trigger a cytokine-cascade phenomenon (Oppenheim and Feldmann, 2001) like those defined for systemic organ systems (Feldmann and Brennan, 2001;Oppenheim and Feldmann, 2001). We believe (Hulse et al, 2004), like others (del Zoppo et al, 2000;Stoll et al, 2000), such cascade behavior also is likely to be important for brain function, including SD. Furthermore like others (Wang et al, 2000a, b), these investigators also suggest that to more fully understand cytokine impact, measurement of targeted cytokine proteins is preferable (when possible) to measurement of related mRNA species.…”

“…Variations between cytokine levels reported here for HOTCs and those in the literature for their in vivo counterparts may be due to differences in animal or sample handling, differences in choice of antibody pairs for assays, differences in laboratory techniques, and of course differences between animal preparations (i.e., whole brain and HOTCs) (for review see Hulse et al, 2004).…”

“…The improved insight needed to further clarify complex mechanisms by which cytokines affect IT can stem from simultaneous measurement of multiple, related targets (del Zoppo et al, 2000;Hulse et al, 2004). The use of RNAse protection assays that include simultaneous measurement of multiple candidate RNA species is one increasingly used experimental strategy (e.g., Dinkel et al, 2003).…”

α2-Adrenoceptor Stimulation Transforms Immune Responses in Neuritis and Blocks Neuritis-Induced Pain

“…Two samples of 50 l of each supernatant were used immediately for cytokine measurement, and the results were averaged for subsequent data analysis. A nine-plex, beadbased rat cytokine immunoassay kit (Bio-Rad, Hercules, CA) was used for simultaneous detection of granulocyte macrophage-colonystimulating factor (GM-CSF), interferon ␥ (IFN␥), IL -10, IL-1␣, IL-1␤, IL-2, IL-4, IL-6, and TNF␣ concentrations, following the manufacturer's instructions and as validated previously (Hulse et al, 2005). Multiwavelength fluorescence and cytokine concentrations were determined with a luminometer (Luminex 100 system; Luminex, Austin, TX) and Bio-Rad software.…”

Ischemic Cardiomyopathy Following Seizure Induction by Domoic Acid

“…47,48 Cytokine concentrations were calculated from a standard curve (detectable range of 2 to 32,000 pg/mL) for rat interleukins IL-1␣ and IL-1␤, tumor necrosis factor-␣ (TNF-␣), and granular monocyte-colony stimulating factor (GM-CSF), using Bio-Plex Manager software (Bio-Rad Laboratories).…”