Millipore Technical Publications |
 | PO- PS1059EN00 | |
Cellular Signaling in EGF and TNFα-stimulated A549 Lung Carcinoma Cells: Phosphoprotein and Cytokine / Chemokine Multiplex Profiling | ||
| Lit No: | PS1059EN00 | |
| Year: | 2009 | |
| Signaling events initiated by extracellular receptor activation lead to the expression and release of cytokines/chemokines. These events are central to cellular and tissue homeostasis, development and functional response. The complexity and number of protein targets involved in such signaling events and cellular responses that cells may undergo require tools that enable multiplex analysis of samples. Here we demonstrate the use of Luminex technology for the multiplexed analysis of cell signaling and cytokine/chemokine expression following stimulation of A549 cells. Tissue culture supernatants and cell lysate samples were collected over a 24-hour time course from TNFα-stimulated cells. These samples were analyzed using MILLIPLEX MAP and MILLIPLEX MAP EpiQuant immunoassays, respectively. Of the 42 cytokines/chemokines investigated, an increased expression level (as compared to vehicle control) was observed for 21 targets in cell culture supernatants for TNFα−treated cells. Of the 105 phosphotyrosine cell signaling targets analyzed, a response was observed in 38 target sites for TNFα- or EGF-treated cells. The magnitude of cytokine/chemokine and phosphotyrosine response varied significantly between targets. For several phosphorylation sites, a decrease in phosphorylation was observed in response to TNFα treatment. Results were obtained using fewer than 288 microplate wells (three-96 well plates), significantly reducing sample and reagent consumption and laboratory time. This approach clearly demonstrates the applicability and utility of coupling intracellular multiplex screening with the measurement of secreted factors — the end-product of many signaling pathways. Click the PDF above for the full document. | |
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