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 5ʹ-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, 4ʹ,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.
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Abbreviations
- ATP:
-
Adenosine 5ʹ-triphosphate
- DAPI:
-
4ʹ,6-Diamidino-2-phenylindole
- BzATP:
-
2ʹ(3ʹ)-O-(4-Benzoylbenzoyl)adenosine-5ʹ-triphosphate tri(triethylammonium)
References
Adinolfi E, Pizzirani C, et al. P2X(7) receptor: death or life? Purinergic Signal. 2005;1(3):219–27.
Bartlett R, Stokes L, et al. The P2X7 receptor channel: recent developments and the use of P2X7 antagonists in models of disease. Pharmacol Rev. 2014;66(3):638–75.
Browne LE, Compan V, et al. P2X7 receptor channels allow direct permeation of nanometer-sized dyes. J Neurosci. 2013;33(8):3557–66.
Cao E, Liao M, et al. TRPV1 structures in distinct conformations reveal activation mechanisms. Nature. 2013;504(7478):113–8.
Chung MK, Guler AD, et al. TRPV1 shows dynamic ionic selectivity during agonist stimulation. Nat Neurosci. 2008;11(5):555–64.
Edelstein, A., N. Amodaj, et al. “Computer control of microscopes using microManager.” Curr Protoc Mol Biol. 2010; Chapter 14: Unit14 20.
Hattori M, Gouaux E. Molecular mechanism of ATP binding and ion channel activation in P2X receptors. Nature. 2012;485(7397):207–12.
Hille, B. (2001). Ion channels of excitable membranes, Sinauer Associates.
Idzko M, Ferrari D, et al. Nucleotide signalling during inflammation. Nature. 2014;509(7500):310–7.
Kawate T, Michel JC, et al. Crystal structure of the ATP-gated P2X(4) ion channel in the closed state. Nature. 2009;460(7255):592–8.
Kepp O, Galluzzi L, et al. Cell death assays for drug discovery. Nat Rev Drug Discov. 2011;10(3):221–37.
Khakh BS, Bao XR, et al. Neuronal P2X transmitter-gated cation channels change their ion selectivity in seconds. Nat Neurosci. 1999;2(4):322–30.
Kuo MT, Chen HH, et al. The roles of copper transporters in cisplatin resistance. Cancer Metastasis Rev. 2007;26(1):71–83.
Liao M, Cao E, et al. Structure of the TRPV1 ion channel determined by electron cryo-microscopy. Nature. 2013;504(7478):107–12.
Munerati M, Cortesi R, et al. Macrophages loaded with doxorubicin by ATP-mediated permeabilization: potential carriers for antitumor therapy. Biochim Biophys Acta. 1994;1224(2):269–76.
North RA. Molecular physiology of P2X receptors. Physiol Rev. 2002;82(4):1013–67.
Penuela S, Gehi R, et al. The biochemistry and function of pannexin channels. Biochim Biophys Acta. 2013;1828(1):15–22.
Schachter J, Motta AP, et al. ATP-induced P2X7-associated uptake of large molecules involves distinct mechanisms for cations and anions in macrophages. J Cell Sci. 2008;121(Pt 19):3261–70.
Schuttelkopf AW, van Aalten DM. PRODRG: a tool for high-throughput crystallography of protein-ligand complexes. Acta Crystallogr D Biol Crystallogr. 2004;60(Pt 8):1355–63.
Virginio C, MacKenzie A, et al. Kinetics of cell lysis, dye uptake and permeability changes in cells expressing the rat P2X7 receptor. J Physiol. 1999a;519(Pt 2):335–46.
Virginio C, MacKenzie A, et al. Pore dilation of neuronal P2X receptor channels. Nat Neurosci. 1999b;2(4):315–21.
Vyklicky L, Novakova-Tousova K, et al. Calcium-dependent desensitization of vanilloid receptor TRPV1: a mechanism possibly involved in analgesia induced by topical application of capsaicin. Physiol Res. 2008;57 Suppl 3:S59–68.
Wiley JS, Chen R, et al. The ATP4- receptor-operated channel (P2Z class) of human lymphocytes allows Ba2+ and ethidium + uptake: inhibition of fluxes by suramin. Arch Biochem Biophys. 1993;305(1):54–60.
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Supplemental Figure 1
No evidence of propidium uptake in P2X7-expressing cells in response to 5 mM ATP. Pooled fluorescence intensity data for propidium in P2X7-expressing HEK293 cells challenged with 5 mM ATP (applied for the duration shown by the black bar). n = 3. (PNG 54 kb)
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Bukhari, M., Burm, H. & Samways, D.S.K. Ion channel-mediated uptake of cationic vital dyes into live cells: a potential source of error when assessing cell viability. Cell Biol Toxicol 32, 363–371 (2016). https://doi.org/10.1007/s10565-016-9344-y
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DOI: https://doi.org/10.1007/s10565-016-9344-y