title: Fluorescence as an alternative to light-scatter gating strategies to identify frozen-thawed cells with flow cytometry.
abstract: Flow cytometry is a key instrument in biological studies, used to identify and analyze cells in suspension. The identification of cells from debris is commonly based on light scatter properties as it has been shown that there is a relationship between forward scattered light and cell volume and this has become common practice in flow cytometry. Cryobiological conditions induce changes in cells that alter their light scatter properties. Cells with membrane damage from freeze-thaw stress produce lower forward scatter signals and may fall below standard forward scatter thresholds. In contrast to light scatter properties that cannot identify damaged cells from debris, fluorescent dyes used in membrane integrity and mitochondrial polarization assays are capable of labeling and discriminating all cells in suspension. Under cryobiological conditions, isolating cell populations is more effectively accomplished by gating on fluorescence rather than light scatter properties. This study shows the limitations of using forward scatter thresholds in flow cytometry to identify and gate cells after exposure to a freeze-thaw protocol and demonstrates the use of fluorescence as an alternative means of identifying and analyzing cells.
authors.affiliation: Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Anthony J F
authors.initials: AJ
authors.lastname: Reardon
authors.affiliation: Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada; Department of Chemical and Materials Engineering, University of Alberta, Edmonton, Alberta, Canada. Electronic address: janet.elliott@ualberta.ca.
authors.forename: Janet A W
authors.initials: JA
authors.lastname: Elliott
authors.affiliation: Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Locksley E
authors.initials: LE
authors.lastname: McGann
mesh_terms: D048429:Cell Size; D002478:Cells, Cultured; D015925:Cryopreservation; D005434:Flow Cytometry / Q000379:methods*; D005453:Fluorescence; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology*; D006801:Humans; D053078:Membrane Potential, Mitochondrial
keywords: Cell gating; Cryobiology; Cryopreservation; Flow cytometry; Fluorescence; Human umbilical vein endothelial cells (HUVEC); Light scattering; Membrane integrity; Mitochondria; Mitochondrial membrane potential