Technical note Amine reactive dyes: An effective tool to discriminate live and dead cells in polychromatic flow cytometry Stephen P. Perfetto, Pratip K. Chattopadhyay, Laurie Lamoreaux, Richard Nguyen, David Ambrozak, Richard A. Koup, Mario Roederer ⁎ Immunology Laboratory, Vaccine Research Center, NIAID, NIH, 40 Convent Dr, Room 5509, Bethesda, MD 20892, United States Received 19 January 2006; accepted 4 April 2006 Available online 19 May 2006 Abstract Membrane-damaged cells caused by either mechanical trauma or through normal biological processes can produce artifacts in immunophenotyping analysis by flow cytometry. Dead cells can nonspecifically bind monoclonal antibody conjugates, potentially leading to erroneous conclusions, particularly when cell frequencies are low. To date, DNA intercalating dyes (Ethidium monoazaide (EMA), Propidium Iodide, 7AAD, etc.) or Annexin V have been commonly used to exclude dead cells; however, each suffer from technical problems. The first issue with such dyes is the dependence on a consistent dead cell source for fluorescence compensation. Another major issue is the stability of the staining; except for EMA, fixation and permeablization used for intracellular staining procedures can cause loss of fluorescence. EMA requires a UV exposure to covalently bond to DNA; while this dye is effective and is not affected by intracellular treatments it is weakly fluorescent. Here we report on the optimization of a new class of viability dyes, the amine reactive viability dyes (ViD) as a dead cell exclusion marker. The inclusion of ViD into the staining panel was found to be simple, reproducible and can have a significant benefit on the accuracy of identifying appropriate cell populations. We show the fluorescence of cells stained with these dyes correlates with traditional dead cell discriminating markers, even after fixation and permeabilization. Amine reactive viability dyes are a powerful tool for fluorescence immunophenotyping experiments. Published by Elsevier B.V. Keywords: Amine reactive dye; Cell viability; Non-specific MAB-conjugate binding 1. Introduction Immune monitoring and vaccine immunogenicity studies often require the measurement of low frequency cell populations. This inevitably leads to questions of sensitivity and reproducibility, since non-specific bind- ing of monoclonal antibody (mAb)-conjugates to dead cells can lead to significant measurement errors (O'Brien and Bolton, 1995; Schmid et al., 1999; Perfetto et al., 2004; Maecker et al., 2005). Viability dyes may be used to exclude dead cells from analysis; intercalating viability dyes enter damaged cells through open membranes and bind DNA. However, the dye may leak out of cells within a short period of time, leading to significant signal loss (Desrues et al., 1989; Costantino et al., 1995; Clarke and Pinder, 1998). This is particularly problematic when permeablization reagents are used to stain intracellular molecules. This problem is avoided when using ethidium monoazide (EMA), which covalently binds to DNA after exposure to ultraviolet Journal of Immunological Methods 313 (2006) 199 – 208 www.elsevier.com/locate/jim ⁎ Corresponding author. Tel.: +1 301 594 8491; fax: +1 301 480 2651. E-mail address: Roederer@nih.gov (M. Roederer). 0022-1759/$ - see front matter. Published by Elsevier B.V. doi:10.1016/j.jim.2006.04.007