A fast and reproducible cell- and 96-well plate-based method for the evaluation of P2X7 receptor activation using YO-PRO-1 fluorescent dye
Main Article Content
Keywords
cytometry, degenerescence, fluorescence, microplate, P2X7 receptor
Abstract
The YO-PRO-1 assay provides a quantitative estimation of P2X7 receptor activation. P2X7 receptor is associated to pathological conditions including infectious, inflammatory, neurological, musculoskeletal disorders, pain and cancer. Most primary cells and cell lines from diverse origin may be used thanks to the ubiquitous distribution of P2X7 receptor. To study the activation of P2X7 receptor by chemicals or biological agents, we established a microplate-based cytometry protocol to accurately and rapidly quantify the activation of P2X7 receptor that leads to the formation of large pores in cell membranes. The YO-PRO-1 assay is based on the ability of cells to incorporate and bind YO-PRO-1 dye to DNA after activation of P2X7 receptor through pore formation. Cells are seeded in 96-well plates and incubated with the compound being tested for the appropriate time. The microplate is then incubated for 10 min with YO-PRO-1 staining solution. After the 10 min staining time, fluorescence signal is read using a microplate reader in 1 min. This procedure is easier and requires less handling steps than flow cytometry. 96-well plate based YO-PRO-1 assay is a reproducible and fast method to study both P2X7 receptor activation by toxic agents at subnecrotic concentrations and P2X7 receptor inhibition by antagonists.
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References
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12. Giannuzzo A, Pedersen SF, Novak I (2015) The P2X7 receptor regulates cell survival, migration and invasion of pancreatic ductal adenocarcinoma cells. Mol Cancer 14: 203.
13. Barros-Barbosa AR, Lobo MG, Ferreirinha F, Correia-de-Sa P, Cordeiro JM (2015) P2X7 receptor activation downmodulates Na(+)-dependent high-affinity GABA and glutamate transport into rat brain cortex synaptosomes. Neuroscience 306: 74-90.
14. Roger S, Pelegrin P, Surprenant A (2008) Facilitation of P2X7 receptor currents and membrane blebbing via constitutive and dynamic calmodulin binding. J Neurosci 28: 6393-6401.
15. Verhoef PA, Estacion M, Schilling W, Dubyak GR (2003) P2X7 receptor-dependent blebbing and the activation of Rho-effector kinases, caspases, and IL-1 beta release. J Immunol 170: 5728-5738.
16. Delarasse C, Gonnord P, Galante M, Auger R, Daniel H, et al. (2009) Neural progenitor cell death is induced by extracellular ATP via ligation of P2X7 receptor. J Neurochem 109: 846-857.
17. Sugiyama T, Oku H, Shibata M, Fukuhara M, Yoshida H, et al. (2010) Involvement of P2X7 receptors in the hypoxia-induced death of rat retinal neurons. Invest Ophthalmol Vis Sci 51: 3236-3243.
18. Zhang X, Zhang M, Laties AM, Mitchell CH (2005) Stimulation of P2X7 receptors elevates Ca2+ and kills retinal ganglion cells. Invest Ophthalmol Vis Sci 46: 2183-2191.
19. North RA (2002) Molecular physiology of P2X receptors. Physiol Rev 82: 1013-1067.
20. Miller CM, Boulter NR, Fuller SJ, Zakrzewski AM, Lees MP, et al. (2011) The role of the P2X(7) receptor in infectious diseases. PLoS Pathog 7: e1002212.
21. Correa G, de ALC, Fernandes-Santos C, Gandini M, Petitinga Paiva F, et al. (2016) The purinergic receptor P2X7 role in control of Dengue virus-2 infection and cytokine/chemokine production in infected human monocytes. Immunobiology.
22. Mortaz E, Adcock IM, Shafei H, Masjedi MR, Folkerts G (2012) Role of P2X7 Receptors in Release of IL-1beta: A Possible Mediator of Pulmonary Inflammation. Tanaffos 11: 6-11.
23. Portales-Cervantes L, Nino-Moreno P, Doniz-Padilla L, Baranda-Candido L, Garcia-Hernandez M, et al. (2010) Expression and function of the P2X(7) purinergic receptor in patients with systemic lupus erythematosus and rheumatoid arthritis. Hum Immunol 71: 818-825.
24. Sanz JM, Falzoni S, Rizzo R, Cipollone F, Zuliani G, et al. (2014) Possible protective role of the 489C>T P2X7R polymorphism in Alzheimer's disease. Exp Gerontol 60: 117-119.
25. Sorge RE, Trang T, Dorfman R, Smith SB, Beggs S, et al. (2012) Genetically determined P2X7 receptor pore formation regulates variability in chronic pain sensitivity. Nat Med 18: 595-599.
26. Agrawal A, Gartland A (2015) P2X7 receptors: role in bone cell formation and function. J Mol Endocrinol 54: R75-88.
27. Liu Z, Liu Y, Xu L, An H, Chang Y, et al. (2015) P2X7 receptor predicts postoperative cancer-specific survival of patients with clear-cell renal cell carcinoma. Cancer Sci 106: 1224-1231.
28. Rassendren F, Buell GN, Virginio C, Collo G, North RA, et al. (1997) The permeabilizing ATP receptor, P2X7. Cloning and expression of a human cDNA. J Biol Chem 272: 5482-5486.
29. Virginio C, Church D, North RA, Surprenant A (1997) Effects of divalent cations, protons and calmidazolium at the rat P2X7 receptor. Neuropharmacology 36: 1285-1294.
30. Idziorek T, Estaquier J, De Bels F, Ameisen JC (1995) YOPRO-1 permits cytofluorometric analysis of programmed cell death (apoptosis) without interfering with cell viability. J Immunol Methods 185: 249-258.
31. Petrou S, Ugur M, Drummond RM, Singer JJ, Walsh JV, Jr. (1997) P2X7 purinoceptor expression in Xenopus oocytes is not sufficient to produce a pore-forming P2Z-like phenotype. FEBS Lett 411: 339-345.
32. Chessell IP, Simon J, Hibell AD, Michel AD, Barnard EA, et al. (1998) Cloning and functional characterisation of the mouse P2X7 receptor. FEBS Lett 439: 26-30.
33. Hibell AD, Kidd EJ, Chessell IP, Humphrey PP, Michel AD (2000) Apparent species differences in the kinetic properties of P2X(7) receptors. Br J Pharmacol 130: 167-173.
34. Aga M, Johnson CJ, Hart AP, Guadarrama AG, Suresh M, et al. (2002) Modulation of monocyte signaling and pore formation in response to agonists of the nucleotide receptor P2X(7). J Leukoc Biol 72: 222-232.
35. Narcisse L, Scemes E, Zhao Y, Lee SC, Brosnan CF (2005) The cytokine IL-1beta transiently enhances P2X7 receptor expression and function in human astrocytes. Glia 49: 245-258.
36. Dutot M, Pouzaud F, Larosche I, Brignole-Baudouin F, Warnet JM, et al. (2006) Fluoroquinolone eye drop-induced cytotoxicity: role of preservative in P2X7 cell death receptor activation and apoptosis. Invest Ophthalmol Vis Sci 47: 2812-2819.
37. Dutot M, Warnet JM, Baudouin C, Rat P (2008) Cytotoxicity of contact lens multipurpose solutions: role of oxidative stress, mitochondrial activity and P2X7 cell death receptor activation. Eur J Pharm Sci 33: 138-145.
38. Dutot M, Liang H, Pauloin T, Brignole-Baudouin F, Baudouin C, et al. (2008) Effects of toxic cellular stresses and divalent cations on the human P2X7 cell death receptor. Mol Vis 14: 889-897.
39. Said T, Dutot M, Christon R, Beaudeux JL, Martin C, et al. (2007) Benefits and side effects of different vegetable oil vectors on apoptosis, oxidative stress, and P2X7 cell death receptor activation. Invest Ophthalmol Vis Sci 48: 5000-5006.
40. Dutot M, Paillet H, Chaumeil C, Warnet JM, Rat P (2009) Severe ocular infections with contact lens: role of multipurpose solutions. Eye (Lond) 23: 470-476.
41. Pauloin T, Dutot M, Warnet JM, Rat P (2008) In vitro modulation of preservative toxicity: high molecular weight hyaluronan decreases apoptosis and oxidative stress induced by benzalkonium chloride. Eur J Pharm Sci 34: 263-273.
42. Pauloin T, Dutot M, Liang H, Chavinier E, Warnet JM, et al. (2009) Corneal protection with high-molecular-weight hyaluronan against in vitro and in vivo sodium lauryl sulfate-induced toxic effects. Cornea 28: 1032-1041.
43. Sigward E, Mignet N, Rat P, Dutot M, Muhamed S, et al. (2013) Formulation and cytotoxicity evaluation of new self-emulsifying multiple W/O/W nanoemulsions. Int J Nanomedicine 8: 611-625.
44. Massicot F, Hache G, David L, Chen D, Leuxe C, et al. (2013) P2X7 Cell Death Receptor Activation and Mitochondrial Impairment in Oxaliplatin-Induced Apoptosis and Neuronal Injury: Cellular Mechanisms and Approach. PLoS One 8: e66830.
45. Wakx A, Regazzetti A, Dargere D, Auzeil N, Gil S, et al. (2016) New in vitro biomarkers to detect toxicity in human placental cells: The example of benzo[A]pyrene. Toxicol In Vitro 32: 76-85.
46. Wakx A, Dutot M, Massicot F, Mascarelli F, Limb GA, et al. (2015) Amyloid beta Peptide Induces Apoptosis Through P2X7 Cell Death Receptor in Retinal Cells: Modulation by Marine Omega-3 Fatty Acid DHA and EPA. Appl Biochem Biotechnol.
47. Allen P, Davies D (2007) Apoptosis Detection by Flow Cytometry. In: Macey MG, editor. Flow Cytometry: Principles and Applications. Totowa, NJ: Humana Press. pp. 147-163.
48. Olivier E, Dutot M, Regazzetti A, Leguillier T, Dargere D, et al. (2016) P2X7-pannexin-1 and amyloid beta-induced oxysterol input in human retinal cell: Role in age-related macular degeneration? Biochimie 127: 70-78.
49. Repetto G, del Peso A, Zurita JL (2008) Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nat Protoc 3: 1125-1131.
50. Slaughter MR, Bugelski PJ, O'Brien PJ (1999) Evaluation of alamar blue reduction for the in vitro assay of hepatocyte toxicity. Toxicol In Vitro 13: 567-569.
51. Hansen J, Bross P (2010) A cellular viability assay to monitor drug toxicity. Methods Mol Biol 648: 303-311.
52. Jiang LH (2009) Inhibition of P2X(7) receptors by divalent cations: old action and new insight. Eur Biophys J 38: 339-346.
2. Szymanski P, Markowicz M, Mikiciuk-Olasik E (2012) Adaptation of high-throughput screening in drug discovery-toxicological screening tests. Int J Mol Sci 13: 427-452.
3. Burnstock G, Knight GE (2004) Cellular distribution and functions of P2 receptor subtypes in different systems. Int Rev Cytol 240: 31-304.
4. Sperlagh B, Vizi ES, Wirkner K, Illes P (2006) P2X7 receptors in the nervous system. Prog Neurobiol 78: 327-346.
5. Wiley JS, Sluyter R, Gu BJ, Stokes L, Fuller SJ (2011) The human P2X7 receptor and its role in innate immunity. Tissue Antigens 78: 321-332.
6. Chang MY, Lu JK, Tian YC, Chen YC, Hung CC, et al. (2011) Inhibition of the P2X7 receptor reduces cystogenesis in PKD. J Am Soc Nephrol 22: 1696-1706.
7. Li CH, Lu XJ, Li MY, Chen J (2015) Cathelicidin modulates the function of monocytes/macrophages via the P2X7 receptor in a teleost, Plecoglossus altivelis. Fish Shellfish Immunol 47: 878-885.
8. Okura D, Horishita T, Ueno S, Yanagihara N, Sudo Y, et al. (2015) Lidocaine preferentially inhibits the function of purinergic P2X7 receptors expressed in Xenopus oocytes. Anesth Analg 120: 597-605.
9. Surprenant A, Rassendren F, Kawashima E, North RA, Buell G (1996) The cytolytic P2Z receptor for extracellular ATP identified as a P2X receptor (P2X7). Science 272: 735-738.
10. Bianchi BR, Lynch KJ, Touma E, Niforatos W, Burgard EC, et al. (1999) Pharmacological characterization of recombinant human and rat P2X receptor subtypes. Eur J Pharmacol 376: 127-138.
11. Zhao H, Zhang X, Dai Z, Feng Y, Li Q, et al. (2016) P2X7 Receptor Suppression Preserves Blood-Brain Barrier through Inhibiting RhoA Activation after Experimental Intracerebral Hemorrhage in Rats. Sci Rep 6: 23286.
12. Giannuzzo A, Pedersen SF, Novak I (2015) The P2X7 receptor regulates cell survival, migration and invasion of pancreatic ductal adenocarcinoma cells. Mol Cancer 14: 203.
13. Barros-Barbosa AR, Lobo MG, Ferreirinha F, Correia-de-Sa P, Cordeiro JM (2015) P2X7 receptor activation downmodulates Na(+)-dependent high-affinity GABA and glutamate transport into rat brain cortex synaptosomes. Neuroscience 306: 74-90.
14. Roger S, Pelegrin P, Surprenant A (2008) Facilitation of P2X7 receptor currents and membrane blebbing via constitutive and dynamic calmodulin binding. J Neurosci 28: 6393-6401.
15. Verhoef PA, Estacion M, Schilling W, Dubyak GR (2003) P2X7 receptor-dependent blebbing and the activation of Rho-effector kinases, caspases, and IL-1 beta release. J Immunol 170: 5728-5738.
16. Delarasse C, Gonnord P, Galante M, Auger R, Daniel H, et al. (2009) Neural progenitor cell death is induced by extracellular ATP via ligation of P2X7 receptor. J Neurochem 109: 846-857.
17. Sugiyama T, Oku H, Shibata M, Fukuhara M, Yoshida H, et al. (2010) Involvement of P2X7 receptors in the hypoxia-induced death of rat retinal neurons. Invest Ophthalmol Vis Sci 51: 3236-3243.
18. Zhang X, Zhang M, Laties AM, Mitchell CH (2005) Stimulation of P2X7 receptors elevates Ca2+ and kills retinal ganglion cells. Invest Ophthalmol Vis Sci 46: 2183-2191.
19. North RA (2002) Molecular physiology of P2X receptors. Physiol Rev 82: 1013-1067.
20. Miller CM, Boulter NR, Fuller SJ, Zakrzewski AM, Lees MP, et al. (2011) The role of the P2X(7) receptor in infectious diseases. PLoS Pathog 7: e1002212.
21. Correa G, de ALC, Fernandes-Santos C, Gandini M, Petitinga Paiva F, et al. (2016) The purinergic receptor P2X7 role in control of Dengue virus-2 infection and cytokine/chemokine production in infected human monocytes. Immunobiology.
22. Mortaz E, Adcock IM, Shafei H, Masjedi MR, Folkerts G (2012) Role of P2X7 Receptors in Release of IL-1beta: A Possible Mediator of Pulmonary Inflammation. Tanaffos 11: 6-11.
23. Portales-Cervantes L, Nino-Moreno P, Doniz-Padilla L, Baranda-Candido L, Garcia-Hernandez M, et al. (2010) Expression and function of the P2X(7) purinergic receptor in patients with systemic lupus erythematosus and rheumatoid arthritis. Hum Immunol 71: 818-825.
24. Sanz JM, Falzoni S, Rizzo R, Cipollone F, Zuliani G, et al. (2014) Possible protective role of the 489C>T P2X7R polymorphism in Alzheimer's disease. Exp Gerontol 60: 117-119.
25. Sorge RE, Trang T, Dorfman R, Smith SB, Beggs S, et al. (2012) Genetically determined P2X7 receptor pore formation regulates variability in chronic pain sensitivity. Nat Med 18: 595-599.
26. Agrawal A, Gartland A (2015) P2X7 receptors: role in bone cell formation and function. J Mol Endocrinol 54: R75-88.
27. Liu Z, Liu Y, Xu L, An H, Chang Y, et al. (2015) P2X7 receptor predicts postoperative cancer-specific survival of patients with clear-cell renal cell carcinoma. Cancer Sci 106: 1224-1231.
28. Rassendren F, Buell GN, Virginio C, Collo G, North RA, et al. (1997) The permeabilizing ATP receptor, P2X7. Cloning and expression of a human cDNA. J Biol Chem 272: 5482-5486.
29. Virginio C, Church D, North RA, Surprenant A (1997) Effects of divalent cations, protons and calmidazolium at the rat P2X7 receptor. Neuropharmacology 36: 1285-1294.
30. Idziorek T, Estaquier J, De Bels F, Ameisen JC (1995) YOPRO-1 permits cytofluorometric analysis of programmed cell death (apoptosis) without interfering with cell viability. J Immunol Methods 185: 249-258.
31. Petrou S, Ugur M, Drummond RM, Singer JJ, Walsh JV, Jr. (1997) P2X7 purinoceptor expression in Xenopus oocytes is not sufficient to produce a pore-forming P2Z-like phenotype. FEBS Lett 411: 339-345.
32. Chessell IP, Simon J, Hibell AD, Michel AD, Barnard EA, et al. (1998) Cloning and functional characterisation of the mouse P2X7 receptor. FEBS Lett 439: 26-30.
33. Hibell AD, Kidd EJ, Chessell IP, Humphrey PP, Michel AD (2000) Apparent species differences in the kinetic properties of P2X(7) receptors. Br J Pharmacol 130: 167-173.
34. Aga M, Johnson CJ, Hart AP, Guadarrama AG, Suresh M, et al. (2002) Modulation of monocyte signaling and pore formation in response to agonists of the nucleotide receptor P2X(7). J Leukoc Biol 72: 222-232.
35. Narcisse L, Scemes E, Zhao Y, Lee SC, Brosnan CF (2005) The cytokine IL-1beta transiently enhances P2X7 receptor expression and function in human astrocytes. Glia 49: 245-258.
36. Dutot M, Pouzaud F, Larosche I, Brignole-Baudouin F, Warnet JM, et al. (2006) Fluoroquinolone eye drop-induced cytotoxicity: role of preservative in P2X7 cell death receptor activation and apoptosis. Invest Ophthalmol Vis Sci 47: 2812-2819.
37. Dutot M, Warnet JM, Baudouin C, Rat P (2008) Cytotoxicity of contact lens multipurpose solutions: role of oxidative stress, mitochondrial activity and P2X7 cell death receptor activation. Eur J Pharm Sci 33: 138-145.
38. Dutot M, Liang H, Pauloin T, Brignole-Baudouin F, Baudouin C, et al. (2008) Effects of toxic cellular stresses and divalent cations on the human P2X7 cell death receptor. Mol Vis 14: 889-897.
39. Said T, Dutot M, Christon R, Beaudeux JL, Martin C, et al. (2007) Benefits and side effects of different vegetable oil vectors on apoptosis, oxidative stress, and P2X7 cell death receptor activation. Invest Ophthalmol Vis Sci 48: 5000-5006.
40. Dutot M, Paillet H, Chaumeil C, Warnet JM, Rat P (2009) Severe ocular infections with contact lens: role of multipurpose solutions. Eye (Lond) 23: 470-476.
41. Pauloin T, Dutot M, Warnet JM, Rat P (2008) In vitro modulation of preservative toxicity: high molecular weight hyaluronan decreases apoptosis and oxidative stress induced by benzalkonium chloride. Eur J Pharm Sci 34: 263-273.
42. Pauloin T, Dutot M, Liang H, Chavinier E, Warnet JM, et al. (2009) Corneal protection with high-molecular-weight hyaluronan against in vitro and in vivo sodium lauryl sulfate-induced toxic effects. Cornea 28: 1032-1041.
43. Sigward E, Mignet N, Rat P, Dutot M, Muhamed S, et al. (2013) Formulation and cytotoxicity evaluation of new self-emulsifying multiple W/O/W nanoemulsions. Int J Nanomedicine 8: 611-625.
44. Massicot F, Hache G, David L, Chen D, Leuxe C, et al. (2013) P2X7 Cell Death Receptor Activation and Mitochondrial Impairment in Oxaliplatin-Induced Apoptosis and Neuronal Injury: Cellular Mechanisms and Approach. PLoS One 8: e66830.
45. Wakx A, Regazzetti A, Dargere D, Auzeil N, Gil S, et al. (2016) New in vitro biomarkers to detect toxicity in human placental cells: The example of benzo[A]pyrene. Toxicol In Vitro 32: 76-85.
46. Wakx A, Dutot M, Massicot F, Mascarelli F, Limb GA, et al. (2015) Amyloid beta Peptide Induces Apoptosis Through P2X7 Cell Death Receptor in Retinal Cells: Modulation by Marine Omega-3 Fatty Acid DHA and EPA. Appl Biochem Biotechnol.
47. Allen P, Davies D (2007) Apoptosis Detection by Flow Cytometry. In: Macey MG, editor. Flow Cytometry: Principles and Applications. Totowa, NJ: Humana Press. pp. 147-163.
48. Olivier E, Dutot M, Regazzetti A, Leguillier T, Dargere D, et al. (2016) P2X7-pannexin-1 and amyloid beta-induced oxysterol input in human retinal cell: Role in age-related macular degeneration? Biochimie 127: 70-78.
49. Repetto G, del Peso A, Zurita JL (2008) Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nat Protoc 3: 1125-1131.
50. Slaughter MR, Bugelski PJ, O'Brien PJ (1999) Evaluation of alamar blue reduction for the in vitro assay of hepatocyte toxicity. Toxicol In Vitro 13: 567-569.
51. Hansen J, Bross P (2010) A cellular viability assay to monitor drug toxicity. Methods Mol Biol 648: 303-311.
52. Jiang LH (2009) Inhibition of P2X(7) receptors by divalent cations: old action and new insight. Eur Biophys J 38: 339-346.
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Jan 20, 2017
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RAT P, OLIVIER E, TANTER C, WAKX A, Dutot M. A fast and reproducible cell- and 96-well plate-based method for the evaluation of P2X7 receptor activation using YO-PRO-1 fluorescent dye. J Biol Methods [Internet]. 2017 Jan. 20 [cited 2024 Apr. 24];4(1):e64. Available from: https://jbmethods.org/index.php/jbm/article/view/136
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