메뉴 건너뛰기




Volumn 36, Issue 8, 2008, Pages 909-921

A simple, novel, procedure for monitoring membrane scrambling and permeability in microparticles, platelets, and leukocytes in whole blood samples

Author keywords

[No Author keywords available]

Indexed keywords

CALCIMYCIN; DYE; ETHIDIUM BROMIDE; HEPARIN; LIPOCORTIN 5; MEMBRANE LIPID; MEROCYANINE; PROPIDIUM IODIDE;

EID: 47149115305     PISSN: 0301472X     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.exphem.2008.03.002     Document Type: Article
Times cited : (14)

References (47)
  • 1
    • 0031036984 scopus 로고    scopus 로고
    • Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death (necrosis)
    • Darzynkiewicz Z., Juan G., Li X., Gorczya W., Murakami T., and Traganos F. Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death (necrosis). Cytometry 27 (1997) 1-20
    • (1997) Cytometry , vol.27 , pp. 1-20
    • Darzynkiewicz, Z.1    Juan, G.2    Li, X.3    Gorczya, W.4    Murakami, T.5    Traganos, F.6
  • 2
    • 0029043888 scopus 로고
    • A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V
    • Vermes I., Haanen C., Steffens-Nakken H., and Reutelingsperger C. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J Immunol Methods 184 (1995) 39-51
    • (1995) J Immunol Methods , vol.184 , pp. 39-51
    • Vermes, I.1    Haanen, C.2    Steffens-Nakken, H.3    Reutelingsperger, C.4
  • 3
    • 0026744185 scopus 로고
    • Clustering of lipid-bound annexin V may explain its anticoagulant effect
    • Andree H.A., Stuart M.C., Hermens W.T., et al. Clustering of lipid-bound annexin V may explain its anticoagulant effect. J Biol Chem 267 (1992) 17907-17912
    • (1992) J Biol Chem , vol.267 , pp. 17907-17912
    • Andree, H.A.1    Stuart, M.C.2    Hermens, W.T.3
  • 4
    • 0032998344 scopus 로고    scopus 로고
    • Optimal storage conditions for preserving granulocyte viability as monitored by Annexin V binding in whole blood
    • Hodge G.L., Flower R., and Han P. Optimal storage conditions for preserving granulocyte viability as monitored by Annexin V binding in whole blood. J Immunol Methods 225 (1999) 27-38
    • (1999) J Immunol Methods , vol.225 , pp. 27-38
    • Hodge, G.L.1    Flower, R.2    Han, P.3
  • 5
    • 0037942894 scopus 로고    scopus 로고
    • Flow cytometric methods for the detection and quantification of apoptosis
    • Buolamwini J.K., and Adjei A.A. (Eds), Humana Press Inc, Totowa, NJ
    • Steensma D.P., Timm M., and Witzig T.E. Flow cytometric methods for the detection and quantification of apoptosis. In: Buolamwini J.K., and Adjei A.A. (Eds). Methods in Molecular Medicine vol 85 (2003), Humana Press Inc, Totowa, NJ 323-332
    • (2003) Methods in Molecular Medicine , vol.85 , pp. 323-332
    • Steensma, D.P.1    Timm, M.2    Witzig, T.E.3
  • 6
    • 0028144891 scopus 로고
    • Effect of fixation on quantification of the expression of leukocyte function-associated surface antigens
    • McCarthy D.A., Macey M.G., Cahill M.R., and Newland A.C. Effect of fixation on quantification of the expression of leukocyte function-associated surface antigens. Cytometry 17 (1994) 39-49
    • (1994) Cytometry , vol.17 , pp. 39-49
    • McCarthy, D.A.1    Macey, M.G.2    Cahill, M.R.3    Newland, A.C.4
  • 7
    • 33644639240 scopus 로고    scopus 로고
    • Paraformaldehyde induces elevation of intracellular calcium and phosphatidylserine externalization in platelets
    • Wong K., Li X., and Ma Y. Paraformaldehyde induces elevation of intracellular calcium and phosphatidylserine externalization in platelets. Thromb Res 117 (2006) 537-542
    • (2006) Thromb Res , vol.117 , pp. 537-542
    • Wong, K.1    Li, X.2    Ma, Y.3
  • 8
    • 0033389934 scopus 로고    scopus 로고
    • Measurement of phosphatidylserine exposure in leukocytes and platelets by whole blood flow cytometry with annexin V
    • Tait J.F., Smith C., and Wood B.L. Measurement of phosphatidylserine exposure in leukocytes and platelets by whole blood flow cytometry with annexin V. Blood Cell Mol Dis 25 (1999) 271-278
    • (1999) Blood Cell Mol Dis , vol.25 , pp. 271-278
    • Tait, J.F.1    Smith, C.2    Wood, B.L.3
  • 9
    • 0026543962 scopus 로고
    • Analysis and discrimination of necrosis and apoptosis (programmed cell death) by multiparametric flow cytometry
    • Dive C., Gregory C.D., Phipps D.J., Evans D.I., Milner A.E., and Wylie A.H. Analysis and discrimination of necrosis and apoptosis (programmed cell death) by multiparametric flow cytometry. Biochim Biophys Acta 1133 (1992) 275-285
    • (1992) Biochim Biophys Acta , vol.1133 , pp. 275-285
    • Dive, C.1    Gregory, C.D.2    Phipps, D.J.3    Evans, D.I.4    Milner, A.E.5    Wylie, A.H.6
  • 10
    • 0028928480 scopus 로고
    • Effects of cell purification methods on CD11b and L-selectin expression as well as the adherence and activation of leucocytes
    • Macey M.G., McCarthy D.A., Vordermeier S., Newland A.C., and Brown K.A. Effects of cell purification methods on CD11b and L-selectin expression as well as the adherence and activation of leucocytes. J Immunol Methods 181 (1995) 211-219
    • (1995) J Immunol Methods , vol.181 , pp. 211-219
    • Macey, M.G.1    McCarthy, D.A.2    Vordermeier, S.3    Newland, A.C.4    Brown, K.A.5
  • 11
    • 18744406663 scopus 로고    scopus 로고
    • Phosphatidylserine content is a more important contributor than transmembrane potential to interactions of merocyanine 540 with lipid bilayers
    • Waczulikova I., Rozalski M., Rievaj J., Nagyova K., Bryszewska M., and Watala C. Phosphatidylserine content is a more important contributor than transmembrane potential to interactions of merocyanine 540 with lipid bilayers. Biochim Biophys Acta 1567 (2002) 176-182
    • (2002) Biochim Biophys Acta , vol.1567 , pp. 176-182
    • Waczulikova, I.1    Rozalski, M.2    Rievaj, J.3    Nagyova, K.4    Bryszewska, M.5    Watala, C.6
  • 12
    • 0036146146 scopus 로고    scopus 로고
    • Interaction of merocyanine 540 with charged membranes
    • Mateasik A., Sikurova L., and Chorvat Jr. D. Interaction of merocyanine 540 with charged membranes. Bioelectrochemistry 55 (2002) 173-175
    • (2002) Bioelectrochemistry , vol.55 , pp. 173-175
    • Mateasik, A.1    Sikurova, L.2    Chorvat Jr., D.3
  • 13
    • 12444274309 scopus 로고    scopus 로고
    • pH, serum proteins and ionic strength influence the uptake of merocyanine 540 by WiDr cells and its interaction with membrane structures
    • Cunderlikova B., Sikurova L., and Moan J. pH, serum proteins and ionic strength influence the uptake of merocyanine 540 by WiDr cells and its interaction with membrane structures. Bioelectrochemistry 59 (2003) 1-10
    • (2003) Bioelectrochemistry , vol.59 , pp. 1-10
    • Cunderlikova, B.1    Sikurova, L.2    Moan, J.3
  • 14
    • 0029077695 scopus 로고
    • Factors affecting the amount and the mode of merocyanine 540 binding to the membrane of human erythrocytes. A comparison with the binding to leukemia cells
    • Lagerberg J.W., Kallen K.J., Haest C.W., VanSteveninck J., and Dubbelman T.M. Factors affecting the amount and the mode of merocyanine 540 binding to the membrane of human erythrocytes. A comparison with the binding to leukemia cells. Biochim Biophys Acta 1235 (1995) 428-436
    • (1995) Biochim Biophys Acta , vol.1235 , pp. 428-436
    • Lagerberg, J.W.1    Kallen, K.J.2    Haest, C.W.3    VanSteveninck, J.4    Dubbelman, T.M.5
  • 15
    • 0023739417 scopus 로고
    • Merocyanine 540 as a flow cytometric probe of membrane lipid organization in leukocytes
    • McEvoy L., Schlegel R.A., Williamson P., and Del Buono B.J. Merocyanine 540 as a flow cytometric probe of membrane lipid organization in leukocytes. J Leukocyte Biol 44 (1988) 337-344
    • (1988) J Leukocyte Biol , vol.44 , pp. 337-344
    • McEvoy, L.1    Schlegel, R.A.2    Williamson, P.3    Del Buono, B.J.4
  • 17
    • 0021113818 scopus 로고
    • Meroocyanne 540, a fluorescent probe sensitive to lipid packing
    • Williamson P., Mattocks K., and Schlegel R.A. Meroocyanne 540, a fluorescent probe sensitive to lipid packing. Biochim Biophys Acta 732 (1983) 387-393
    • (1983) Biochim Biophys Acta , vol.732 , pp. 387-393
    • Williamson, P.1    Mattocks, K.2    Schlegel, R.A.3
  • 18
    • 0022636912 scopus 로고
    • Alterations in plasma membrane lipid organization during lymphocyte differentiation
    • Del Buono B.J., Williamson P.L., and Schlegel R.A. Alterations in plasma membrane lipid organization during lymphocyte differentiation. J Cell Physiol 126 (1986) 379-388
    • (1986) J Cell Physiol , vol.126 , pp. 379-388
    • Del Buono, B.J.1    Williamson, P.L.2    Schlegel, R.A.3
  • 19
    • 0022569754 scopus 로고
    • Changes in the organization of membrane lipids during human platelet activation. Study by fluorescent and freeze-fracture cytochemistry
    • Lupu F., Calb M., Scurei C., and Simionescu N. Changes in the organization of membrane lipids during human platelet activation. Study by fluorescent and freeze-fracture cytochemistry. Lab Invest 54 (1986) 136-145
    • (1986) Lab Invest , vol.54 , pp. 136-145
    • Lupu, F.1    Calb, M.2    Scurei, C.3    Simionescu, N.4
  • 20
    • 0026508561 scopus 로고
    • Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages
    • Fadok V.A., Voelker D.R., Campbell P.A., Cohen J.J., Bratton D.L., and Henson P.M. Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J Immunol 148 (1992) 2207-2216
    • (1992) J Immunol , vol.148 , pp. 2207-2216
    • Fadok, V.A.1    Voelker, D.R.2    Campbell, P.A.3    Cohen, J.J.4    Bratton, D.L.5    Henson, P.M.6
  • 21
    • 0027209579 scopus 로고
    • Increased membrane permeability of apoptotic thymocytes: a flow cytometric study
    • Ormerod M.G., Sun X.M., Snowden R.T., Davies R., Fearnhead H., and Cohen G.M. Increased membrane permeability of apoptotic thymocytes: a flow cytometric study. Cytometry 14 (1993) 595-602
    • (1993) Cytometry , vol.14 , pp. 595-602
    • Ormerod, M.G.1    Sun, X.M.2    Snowden, R.T.3    Davies, R.4    Fearnhead, H.5    Cohen, G.M.6
  • 22
    • 0037410195 scopus 로고    scopus 로고
    • Platelet apoptosis in stored platelet concentrates and other models
    • Leytin V., and Freedman J. Platelet apoptosis in stored platelet concentrates and other models. Transfus Apher Sci 28 (2003) 285-295
    • (2003) Transfus Apher Sci , vol.28 , pp. 285-295
    • Leytin, V.1    Freedman, J.2
  • 23
    • 0034142375 scopus 로고    scopus 로고
    • Annexin V binds to viable B cells and colocalizes with a marker of lipid rafts upon B cell receptor activation
    • Dillon S.R., Mancini M., Rosen A., and Schlissel M.S. Annexin V binds to viable B cells and colocalizes with a marker of lipid rafts upon B cell receptor activation. J Immunol 164 (2000) 1322-1332
    • (2000) J Immunol , vol.164 , pp. 1322-1332
    • Dillon, S.R.1    Mancini, M.2    Rosen, A.3    Schlissel, M.S.4
  • 24
    • 0036498701 scopus 로고    scopus 로고
    • Versatility of merocyanine 540 for the flow cytometric detection of apoptosis in human and murine cells
    • Laakko T., King L., and Fraker P. Versatility of merocyanine 540 for the flow cytometric detection of apoptosis in human and murine cells. J Immunol Methods 261 (2002) 129-139
    • (2002) J Immunol Methods , vol.261 , pp. 129-139
    • Laakko, T.1    King, L.2    Fraker, P.3
  • 25
    • 0028274792 scopus 로고
    • Decreased membrane phospholipid packing and decreased cell size precede DNA cleavage in mature mouse B cell apoptosis
    • Mower Jr. D.A., Peckham D.W., Illera V.A., Fishbaugh J.K., Stunz L.L., and Ashman R.F. Decreased membrane phospholipid packing and decreased cell size precede DNA cleavage in mature mouse B cell apoptosis. J Immunol 152 (1994) 4832-4842
    • (1994) J Immunol , vol.152 , pp. 4832-4842
    • Mower Jr., D.A.1    Peckham, D.W.2    Illera, V.A.3    Fishbaugh, J.K.4    Stunz, L.L.5    Ashman, R.F.6
  • 26
    • 0036273871 scopus 로고    scopus 로고
    • Evaluation of the anticoagulants EDTA and citrate, theophylline, adenosine, and dipyridamole (CTAD) for assessing platelet activation on the ADVIA 120 hematology system
    • Macey M., Azam U., McCarthy D.A., et al. Evaluation of the anticoagulants EDTA and citrate, theophylline, adenosine, and dipyridamole (CTAD) for assessing platelet activation on the ADVIA 120 hematology system. Clin Chem 48 (2002) 891-899
    • (2002) Clin Chem , vol.48 , pp. 891-899
    • Macey, M.1    Azam, U.2    McCarthy, D.A.3
  • 27
    • 1442325409 scopus 로고    scopus 로고
    • Ethylenediaminetetraacetic acid plus citrate-theophylline-adenosine-dipyridamole (EDTA-CTAD): a novel anticoagulant for the flow cytometric assessment of platelet and neutrophil activation ex vivo in whole blood
    • Macey M.G., McCarthy D.A., Azam U., Milne T., Golledge P., and Newland A. Ethylenediaminetetraacetic acid plus citrate-theophylline-adenosine-dipyridamole (EDTA-CTAD): a novel anticoagulant for the flow cytometric assessment of platelet and neutrophil activation ex vivo in whole blood. Cytometry B Clin Cytom 52B (2003) 30-40
    • (2003) Cytometry B Clin Cytom , vol.52 B , pp. 30-40
    • Macey, M.G.1    McCarthy, D.A.2    Azam, U.3    Milne, T.4    Golledge, P.5    Newland, A.6
  • 29
    • 0034653120 scopus 로고    scopus 로고
    • Low-affinity kainate receptor agonists induce insult-dependent apoptosis and necrosis in cultured murine cortical neurons
    • Moldrich R.X., Beart P.M., Pascoe C.J., and Cheung N.S. Low-affinity kainate receptor agonists induce insult-dependent apoptosis and necrosis in cultured murine cortical neurons. J Neurosci Res 59 (2000) 788-796
    • (2000) J Neurosci Res , vol.59 , pp. 788-796
    • Moldrich, R.X.1    Beart, P.M.2    Pascoe, C.J.3    Cheung, N.S.4
  • 30
    • 0035368620 scopus 로고    scopus 로고
    • Fluorescence microplate-based assay for tumor necrosis factor activity using SYTOX Green stain
    • Jones L.J., and Singer V.L. Fluorescence microplate-based assay for tumor necrosis factor activity using SYTOX Green stain. Anal Biochem 293 (2001) 8-15
    • (2001) Anal Biochem , vol.293 , pp. 8-15
    • Jones, L.J.1    Singer, V.L.2
  • 31
    • 0030958619 scopus 로고    scopus 로고
    • Bacterial viability and antibiotic susceptibility testing with SYTOX green nucleic acid stain
    • Roth B.L., Poot M., Yue S.T., and Millard P.J. Bacterial viability and antibiotic susceptibility testing with SYTOX green nucleic acid stain. Appl Environ Microb 63 (1997) 2421-2431
    • (1997) Appl Environ Microb , vol.63 , pp. 2421-2431
    • Roth, B.L.1    Poot, M.2    Yue, S.T.3    Millard, P.J.4
  • 32
    • 33748514572 scopus 로고    scopus 로고
    • Flow cytometric evaluation of platelet activation in chronic autoimmune thrombocytopenia
    • Panzer S., Hocker L., Vormttag R., et al. Flow cytometric evaluation of platelet activation in chronic autoimmune thrombocytopenia. Pediatr Blood Cancer 47 (2006) 694-696
    • (2006) Pediatr Blood Cancer , vol.47 , pp. 694-696
    • Panzer, S.1    Hocker, L.2    Vormttag, R.3
  • 33
    • 0027203387 scopus 로고
    • A simple flow cytometric procedure for the determination of surface antigens on unfixed leucocytes in whole blood
    • McCarthy D.A., and Macey M.G. A simple flow cytometric procedure for the determination of surface antigens on unfixed leucocytes in whole blood. J Immunol Methods 163 (1993) 155-160
    • (1993) J Immunol Methods , vol.163 , pp. 155-160
    • McCarthy, D.A.1    Macey, M.G.2
  • 34
    • 0029946804 scopus 로고    scopus 로고
    • Flow cytometric analysis of the early phases of apoptosis by cellular and nuclear techniques
    • Ferlini C., Di Cesare S., Rainaldi G., et al. Flow cytometric analysis of the early phases of apoptosis by cellular and nuclear techniques. Cytometry 24 (1996) 106-115
    • (1996) Cytometry , vol.24 , pp. 106-115
    • Ferlini, C.1    Di Cesare, S.2    Rainaldi, G.3
  • 35
    • 0036454440 scopus 로고    scopus 로고
    • Etoposide-mediated deregulation of the G2M checkpoint in myeloid leukaemic cell lines results in loss of cell survival
    • Higginbottom K., Cummings M., Newland A.C., and Allen P.D. Etoposide-mediated deregulation of the G2M checkpoint in myeloid leukaemic cell lines results in loss of cell survival. Br J Haematol 119 (2002) 956-964
    • (2002) Br J Haematol , vol.119 , pp. 956-964
    • Higginbottom, K.1    Cummings, M.2    Newland, A.C.3    Allen, P.D.4
  • 37
    • 0036835719 scopus 로고    scopus 로고
    • Merocyanine 540 as a fluorescent probe of altered membrane phospholipid asymmetry in activated whole blood platelets
    • Watala C., Waczulikova I., Wieclawska B., et al. Merocyanine 540 as a fluorescent probe of altered membrane phospholipid asymmetry in activated whole blood platelets. Cytometry 49 (2002) 119-133
    • (2002) Cytometry , vol.49 , pp. 119-133
    • Watala, C.1    Waczulikova, I.2    Wieclawska, B.3
  • 38
    • 0034840308 scopus 로고    scopus 로고
    • Different orders for acquisition of apoptotic characteristics by leukocytes
    • Hess K.L., Johnson J.D., and Cook-Mills J.M. Different orders for acquisition of apoptotic characteristics by leukocytes. J Leukocyte Biol 70 (2001) 405-412
    • (2001) J Leukocyte Biol , vol.70 , pp. 405-412
    • Hess, K.L.1    Johnson, J.D.2    Cook-Mills, J.M.3
  • 39
    • 0030745869 scopus 로고    scopus 로고
    • Correlated flow cytometric analysis of terminal events in apoptosis reveals the absence of some changes in some model systems
    • Frey T. Correlated flow cytometric analysis of terminal events in apoptosis reveals the absence of some changes in some model systems. Cytometry 2 (1997) 253-263
    • (1997) Cytometry , vol.2 , pp. 253-263
    • Frey, T.1
  • 40
    • 0032979606 scopus 로고    scopus 로고
    • Spontaneous ex vivo apoptosis of peripheral blood mononuclear cells in patients with head and neck cancer
    • Saito T., Dworacki G., Gooding W., Johnson J.T., and Whiteside T.L. Spontaneous ex vivo apoptosis of peripheral blood mononuclear cells in patients with head and neck cancer. Clin Cancer Res 5 (1999) 329-334
    • (1999) Clin Cancer Res , vol.5 , pp. 329-334
    • Saito, T.1    Dworacki, G.2    Gooding, W.3    Johnson, J.T.4    Whiteside, T.L.5
  • 41
    • 33645551730 scopus 로고    scopus 로고
    • Circulating platelet-derived microparticles in systemic lupus erythematosus. Association with increased thrombin generation and procoagulant state
    • Pereira J., Alfaro G., Goycoolea M., et al. Circulating platelet-derived microparticles in systemic lupus erythematosus. Association with increased thrombin generation and procoagulant state. Thromb Haemost 95 (2006) 94-99
    • (2006) Thromb Haemost , vol.95 , pp. 94-99
    • Pereira, J.1    Alfaro, G.2    Goycoolea, M.3
  • 42
    • 0034863137 scopus 로고    scopus 로고
    • Platelet activation: assessment and quantification
    • Kamath S., Blann A.D., and Lip G.Y.H. Platelet activation: assessment and quantification. Eur Heart J 22 (2001) 1561-1571
    • (2001) Eur Heart J , vol.22 , pp. 1561-1571
    • Kamath, S.1    Blann, A.D.2    Lip, G.Y.H.3
  • 43
    • 8744244550 scopus 로고    scopus 로고
    • Procoagulant potential of platelet alpha granules
    • Polasek J. Procoagulant potential of platelet alpha granules. Platelets 15 (2004) 403-407
    • (2004) Platelets , vol.15 , pp. 403-407
    • Polasek, J.1
  • 44
    • 0033373546 scopus 로고    scopus 로고
    • Procoagulant expression in platelets and defects leading to clinical disorders
    • Solum N.O. Procoagulant expression in platelets and defects leading to clinical disorders. Arterioscler Thromb Vasc Biol 19 (1999) 2841-2846
    • (1999) Arterioscler Thromb Vasc Biol , vol.19 , pp. 2841-2846
    • Solum, N.O.1
  • 45
    • 0014109162 scopus 로고
    • Lipids of human leukocytes: relation to cell type
    • Gottfried E.L. Lipids of human leukocytes: relation to cell type. J Lipid Res 8 (1967) 321-327
    • (1967) J Lipid Res , vol.8 , pp. 321-327
    • Gottfried, E.L.1
  • 46
    • 0033799341 scopus 로고    scopus 로고
    • Prostasomes inhibit the NADPH oxidase activity of human neutrophils
    • Saez F., Motta C., Boucher D., and Grizard G. Prostasomes inhibit the NADPH oxidase activity of human neutrophils. Mol Hum Reprod 6 (2000) 883-891
    • (2000) Mol Hum Reprod , vol.6 , pp. 883-891
    • Saez, F.1    Motta, C.2    Boucher, D.3    Grizard, G.4
  • 47
    • 33644699560 scopus 로고    scopus 로고
    • Usefulness of a triple fluorochrome combination Merocyanine 540/Yo-Pro 1/Hoechst 33342 in assessing membrane stability of viable frozen-thawed spermatozoa from Estonian Holstein AI bulls
    • Hallap T., Nagy S., Jaakma U., Johannisson A., and Rodriguez-Martinez H. Usefulness of a triple fluorochrome combination Merocyanine 540/Yo-Pro 1/Hoechst 33342 in assessing membrane stability of viable frozen-thawed spermatozoa from Estonian Holstein AI bulls. Theriogenology 65 (2006) 1122-1136
    • (2006) Theriogenology , vol.65 , pp. 1122-1136
    • Hallap, T.1    Nagy, S.2    Jaakma, U.3    Johannisson, A.4    Rodriguez-Martinez, H.5


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.