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Amine reactive dyes: an effective tool to discriminate live and dead cells in polychromatic flow cytometry
Stephen P Perfetto1, Pratip K Chattopadhyay, Laurie Lamoreaux
1Immunology Laboratory, Vaccine Research Center, NIAID, NIH, 40 Convent Dr, Room 5509, Bethesda, MD 20892, USA.
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.
Insights
New amine-reactive viability dyes (ViD) offer a simple and reproducible method for excluding dead cells in flow cytometry. These dyes improve immunophenotyping accuracy, even after fixation and permeabilization, overcoming limitations of traditional viability markers.
Area of Science:
- * Cellular Biology
- * Immunology
- * Biotechnology
Background:
- * Membrane-damaged cells in flow cytometry can cause artifacts by non-specifically binding antibodies.
- * Traditional dead cell exclusion markers like DNA intercalating dyes and Annexin V have limitations, including fluorescence compensation issues and instability during fixation/permeabilization.
- * Ethidium monoazaide (EMA) is stable but weakly fluorescent and requires UV activation.
Purpose of the Study:
- * To optimize and validate a new class of amine-reactive viability dyes (ViD) as dead cell exclusion markers for flow cytometry.
- * To assess the performance of ViD in improving the accuracy of immunophenotyping.
- * To evaluate the stability and compatibility of ViD with fixation and permeabilization procedures.
Main Methods:
- * Optimization of amine-reactive viability dyes (ViD) for use as dead cell exclusion markers.
- * Staining panels were designed to include ViD alongside traditional markers.
- * Comparison of ViD fluorescence with traditional dead cell discriminating markers, including post-fixation and permeabilization.
Main Results:
- * Amine-reactive viability dyes (ViD) provide a simple and reproducible method for dead cell exclusion.
- * ViD staining fluorescence correlates with traditional dead cell markers.
- * ViD fluorescence remains stable after fixation and permeabilization, unlike some other viability dyes.
Conclusions:
- * Amine-reactive viability dyes (ViD) are a powerful tool for enhancing accuracy in fluorescence immunophenotyping experiments.
- * ViD offers a stable and reliable alternative to existing dead cell exclusion methods.
- * The use of ViD simplifies the process and improves the identification of specific cell populations.
