Abstract 157: Optimizing the flow cytometric analysis of cell signaling in heterogeneous samples

• Published in: Cancer research (Chicago, Ill.) Ill.), 2012-04, Vol.72 (8_Supplement), p.157-157
• Main Authors: Gao, Guojian, Coveney, Christopher, Wang, Xiao, Wu, Xiaowei, Wei, Ai-Li, Ji, Xiang-dong, Anderson, Lori, Kotecha, Nikesh, Rohrer, Jurg, O'Donnell, Erika
• Format: Article
• Language: English
• ISSN: 0008-5472; 1538-7445
• DOI: 10.1158/1538-7445.AM2012-157

Unlike conventional biochemical methods for cell signaling analysis, analyzing protein levels by flow cytometry allows measurements to be made at the single-cell level. Multiparameter flow cytometry provides a powerful tool for the analysis of protein expression and phosphorylation status within complex populations, such as those found in lymphoid tissues or blood. However, different intracellular and cell surface proteins often require different conditions for optimal detection, creating challenges for analyzing multiple protein types in a single sample. To facilitate the design of multiparameter staining panels for analysis of intracellular and cell surface proteins, we tested over 250 fluorescently conjugated antibodies in cells fixed and permeabilized under various conditions. Multiple fluorescent conjugates of each antibody were tested in human PBMCs, human whole blood, mouse spleen, and/or mouse bone marrow following fixation with BD Phosflow™ Lyse/Fix or BD Cytofix™ Fixation Buffer and permeabilization with one of the four BD Phosflow™ Perm buffers. These studies demonstrated that certain intracellular epitopes required harsh permeabilization conditions for optimal detection, whereas others were detectable under milder conditions. Surface marker stains often required optimization, particularly under harsh permeabilization conditions. Factors such as antibody clone, fluorophore, and concentration had a considerable impact on staining resolution, and alternative staining protocols were required for optimal staining of some surface markers. To facilitate the optimization of fixation, permeabilization, and staining conditions for multiparameter flow cytometry, experimental data and protocols from this study are available through an interactive database freely accessible at www.cytobank.org/facselect. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 157. doi:1538-7445.AM2012-157