Abstract 323: A rapid flow cytometry method for analyzing NF-κB activation and translocation in whole blood
Authors: George C. Brittain, Sergei Gulnik
Journal: Cancer Research (2017)

Abstract Background: The end point of most cell-signaling cascades is the translocation of protein complexes into the nucleus to modify transcriptional activity. For this reason, the analysis of cytoplasmic vs. nuclear localization provides important insights into cell activation. However, assessing nuclear translocation in complex cell mixtures, such as whole blood, is a difficult task. Leukocytes are a rare event in blood, with many subpopulations, requiring the cells to first be concentrated, sorted, rested and often expanded prior to further molecular analyses. This process can take weeks, and questions regarding physiological relevance arise from even the first preparatory steps. Here, we present a buffer-based system to lyse RBCs and differentially stain leukocytes in order to enable the quantitative analysis of cytoplasmic vs. nuclear localization in endogenous cells by flow cytometry. We demonstrate the use of this system by analyzing the activation of NF-κB signaling in whole-blood monocytes following lipopolysaccharide (LPS) stimulation. Methods: The main components of the Whole Blood Nuclear Localization Kit are two buffers: Buffer 1 permeabilizes the cytoplasmic membrane, leaving the nuclear membrane intact, while Buffer 2 permeabilizes both the cytoplasmic and nuclear membranes. Briefly, blood was treated for different times with LPS, fixed for 10 min, split into 2 fractions, and then one was lysed for 30 min with Buffer 1 and the other with Buffer 2. Each fraction was washed, stained and acquired on a CytoFLEX-S flow cytometer (Beckman Coulter, Brea, CA). The cytoplasmic and nuclear signals were calculated using the compensated results from the two tubes. Blood was collected onsite daily from healthy, consenting human adults. Results: Following LPS stimulation, nuclear p50 and RelA increased maximally in monocytes by 10 min. Phospho-RelA S536 was maximal by 5 min mostly in the cytoplasm, while phospho-RelA S529 was maximal by 10 min in the nucleus. IκBα degraded maximally by 10 min. Additional kinase signaling will be presented, as well as controls to demonstrate the partitioning efficiency. Conclusions: This system is very fast and user friendly, and can help to improve our understanding of normal and aberrant signaling in blood, while also saving researchers time and resources compared to conventional procedures, e.g., purifying leukocytes and performing Western blots. It provides clear delineation of the nucleus with minimal perinuclear staining, and can even be used to improve the quantitation of nuclear signals from 2D microscopic images. Finally, this system can enable research where blood volumes are limited, such as with rare and precious samples and small-animal studies. ***This kit and the CytoFLEX-S are for Research Use Only. Beckman Coulter and the Beckman Coulter product and service marks mentioned herein are trademarks or registered trademarks of Beckman Coulter, Inc. in the United States and other countries.*** Citation Format: George C. Brittain, Sergei Gulnik. A rapid flow cytometry method for analyzing NF-κB activation and translocation in whole blood [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 323. doi:10.1158/1538-7445.AM2017-323