title: Amine reactive dyes: an effective tool to discriminate live and dead cells in polychromatic flow cytometry.
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.
authors.affiliation: Immunology Laboratory, Vaccine Research Center, NIAID, NIH, 40 Convent Dr, Room 5509, Bethesda, MD 20892, USA.
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Perfetto
authors.forename: Pratip K
authors.initials: PK
authors.lastname: Chattopadhyay
authors.forename: Laurie
authors.initials: L
authors.lastname: Lamoreaux
authors.forename: Richard
authors.initials: R
authors.lastname: Nguyen
authors.forename: David
authors.initials: D
authors.lastname: Ambrozak
authors.forename: Richard A
authors.initials: RA
authors.lastname: Koup
authors.forename: Mario
authors.initials: M
authors.lastname: Roederer
mesh_terms: D000588:Amines / Q000737:chemistry*; D017302:Annexins / Q000737:chemistry; D015703:Antigens, CD / Q000032:analysis; D017209:Apoptosis; D001386:Azides / Q000737:chemistry; D002470:Cell Survival; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000737:chemistry / Q000166:cytology; D008863:Microspheres; D015203:Reproducibility of Results; D013194:Staining and Labeling / Q000379:methods*; D017186:Titrimetry
chemical_list: D000588:Amines; D017302:Annexins; D015703:Antigens, CD; D001386:Azides; D005456:Fluorescent Dyes; C014535:8-azidoethidium; D007371:Interferon-gamma