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Rapid Image-based Cytometry for Comparison of Fluorescent Viability Staining Methods

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Abstract

The ability to accurately measure cell viability is important for any cell-based research. Traditionally, viability measurements have been performed using trypan blue exclusion method on hemacytometer, which allowed researchers to visually distinguish viable from nonviable cells. However, the trypan blue method is often limited to only cell lines or primary cells that have been rigorously purified. In the recent years, small desktop image-based cell counters have been developed for rapid cell concentration and viability measurement due to advances in imaging and optics technologies as well as novel fluorescent stains. In this work, we employed the Cellometer image-based cytometer to demonstrate the ability to simplify viability detection compared to the current methods. We compared various fluorescence viability detection methods using single- or dual-staining technique. Single-staining method using nucleic acid stains including ethidium bromide, propidium iodide, 7AAD, DAPI, Sytox Green and Sytox Red, and enzymatic stains including CFDA and Calcein AM were performed. All stains produced comparable results to trypan blue exclusion method for cell line samples. Dual-staining method using AO/PI, CFDA/PI, Calcein AM/PI and Hoechst 33342/PI that enumerates viable and non-viable cells was tested on primary cell samples with high debris contents. This method allowed exclusion of cellular debris and non-nucleated cells from analysis, which can eliminate the need to perform purification step during sample preparation, and improves the efficiency of viability detection method. Overall, these image-based fluorescent cell counters can simplify assay procedures as well as capture images for visual confirmation.

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Acknowledgments

The authors would like to thank Professor Xuemei Zhong at Boston University Medical Center (Boston, MA) for her kind gift of mouse splenocytes and PBMCs.

Conflict of Interest

The authors, LLC, BDP, and NL declare competing financial interests, and the work performed in this manuscript is for reporting on product performance of Nexcelom Bioscience, LLC. The performance of the instrumentation has been compared to standard approaches currently used in the biomedical research institutions.

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Authors and Affiliations

  1. Department of Technology R&D, Nexcelom Bioscience LLC, 360 Merrimack St. Building 9, Lawrence, MA, 01843, USA

    Leo L. Chan, Alisha R. Wilkinson & Benjamin D. Paradis

  2. Department of Applications, Nexcelom Bioscience LLC, Lawrence, MA, 01843, USA

    Ning Lai

  3. Department of Biology, Merrimack College, North Andover, MA, 01845, USA

    Alisha R. Wilkinson

  4. Center for Biotechnology and Biomedical Sciences, Merrimack College, North Andover, MA, 01845, USA

    Leo L. Chan, Alisha R. Wilkinson & Benjamin D. Paradis

Authors
  1. Leo L. Chan
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  2. Alisha R. Wilkinson
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  3. Benjamin D. Paradis
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  4. Ning Lai
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Corresponding author

Correspondence to Leo L. Chan.

Additional information

Alisha R. Wilkinson and Benjamin D. Paradis contributed equally in this manuscript.

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Chan, L.L., Wilkinson, A.R., Paradis, B.D. et al. Rapid Image-based Cytometry for Comparison of Fluorescent Viability Staining Methods. J Fluoresc 22, 1301–1311 (2012). https://doi.org/10.1007/s10895-012-1072-y

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  • DOI: https://doi.org/10.1007/s10895-012-1072-y

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