title: Ion channel-mediated uptake of cationic vital dyes into live cells: a potential source of error when assessing cell viability.
abstract: Ionic "vital dyes" are commonly used to assess cell viability based on the idea that their permeation is contingent on a loss of membrane integrity. However, the possibility that dye entry is conducted into live cells by endogenous membrane transporters must be recognized and controlled for. Several cation-selective plasma membrane-localized ion channels, including the adenosine 5'-triphosphate (ATP)-gated P2X receptors, have been reported to conduct entry of the DNA-binding fluorescence dye, YO-PRO-1, into live cells. Extracellular ATP often becomes elevated as a result of release from dying cells, and so it is possible that activation of P2X channels on neighboring live cells could lead to exaggerated estimation of cytotoxicity. Here, we screened a number of fluorescent vital dyes for ion channel-mediated uptake in HEK293 cells expressing recombinant P2X2, P2X7, or TRPV1 channels. Our data shows that activation of all three channels caused substantial uptake and nuclear accumulation of YO-PRO-1, 4',6-diamidino-2-phenylindole (DAPI), and Hoechst 33258 into transfected cells and did so well within the time period usually used for incubation of cells with vital dyes. In contrast, channel activation in the presence of propidium iodide and SYTOX Green caused no measurable uptake and accumulation during a 20-min exposure, suggesting that these dyes are not likely to exhibit measurable uptake through these particular ion channels during a conventional cell viability assay. Caution is encouraged when choosing and employing cationic dyes for the purpose of cell viability assessment, particularly when there is a likelihood of cells expressing ion channels permeable to large ions.
authors.affiliation: Department of Biology, Clarkson University, 177 Science Center, Box 5805, Potsdam, NY, 13699-5805, USA.
authors.forename: Maurish
authors.initials: M
authors.lastname: Bukhari
authors.affiliation: School of Medicine and Dentistry, University of Rochester Medical Center, 601 Elmwood Ave, Rochester, NY, USA.
authors.forename: Hayley
authors.initials: H
authors.lastname: Burm
authors.affiliation: Department of Biology, Clarkson University, 177 Science Center, Box 5805, Potsdam, NY, 13699-5805, USA. dsamway@clarkson.edu.
authors.forename: Damien S K
authors.initials: DS
authors.lastname: Samways
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001583:Benzoxazoles / Q000493:pharmacokinetics; D002412:Cations / Q000493:pharmacokinetics; D002470:Cell Survival / Q000502:physiology; D005456:Fluorescent Dyes / Q000493:pharmacokinetics*; D057809:HEK293 Cells; D006801:Humans; D007211:Indoles / Q000378:metabolism; D007473:Ion Channels / Q000378:metabolism*; D011806:Quinolinium Compounds / Q000493:pharmacokinetics*; D058469:Receptors, Purinergic P2X / Q000378:metabolism; D058476:Receptors, Purinergic P2X2 / Q000378:metabolism; D058486:Receptors, Purinergic P2X7 / Q000378:metabolism; D050916:TRPV Cation Channels / Q000378:metabolism
chemical_list: D001583:Benzoxazoles; D002412:Cations; D005456:Fluorescent Dyes; D007211:Indoles; D007473:Ion Channels; C546468:P2RX7 protein, human; D011806:Quinolinium Compounds; D058469:Receptors, Purinergic P2X; D058476:Receptors, Purinergic P2X2; D058486:Receptors, Purinergic P2X7; D050916:TRPV Cation Channels; C494482:TRPV1 protein, human; C089813:YO-PRO 1; C007293:DAPI; D000255:Adenosine Triphosphate
keywords: Cell death; Cell viability; Ion channel; Propidium iodide; Vital dye; YO-PRO-1