Automating Sample Preparation For Single-Cell Protein Phosphorylation Analysis By Flow Cytometry

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Protein phosphorylation plays essential roles in regulating cell proliferation, differentiation and other cellular processes. Aberrant regulation of protein kinases, can drive malignancy through inappropriate cell growth and survival signals. Understanding the phosphorylation status of specific cellular proteins in cancer has proven valuable in developing targeted therapies and assays to identify specific drug responses in individual cancer patients. The ability to analyze cells at the single cell level allows flow cytometry to measure protein phosphorylation in rare subsets of cells in complex populations such as blood or bone marrow specimens. However, sample preparation for protein phosphorylation analysis by flow cytometry can be technically challenging, time-consuming, and subject to error when performed manually. Furthermore, the procedure demands strict control of experimental conditions such as time and temperature due to the dynamic nature of protein phosphorylation. In this report, we describe an automated sample preparation method using a @Biomek® NXP Span-8 work-station to monitor the phosphorylation status of ERK and p38 MAPK in human peripheral blood monocytes. The method allows automation of sample preparation steps, including 1) ERK/p38 MAPK stimulation, 2) fixation, 3) erythrocyte lysis, 4) cell permeabilization, and, 5) staining with phospho-specific and cell surface marker antibodies. Using this approach, we were able to observe p38 MAPK and ERK activation in CD14+ monocytes following lipopolysaccharide (LPS) or phorbol-12-myristate-13 acetate (PMA) treatment. The automated sample preparation workstation is capable of processing 48 samples in approximately 2.5 hours. Note: *Method cited Not for Clinical purposes. @For Laboratory Use Only; not for use in diagnostic procedures. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-230.