JP2002122593A - Method for analyzing peroxidation of cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for analyzing peroxidation of a cell, especially peroxidation of the lipid of a cell membrane. SOLUTION: Diphenyl-1-pyrenylphosphine(DPPP) is provided to a cell and fluorescence of diphenyl-1-pyrenylphosphineoxide (DPPP=0) is measured thus analyzing peroxidation of the cell.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for analyzing peroxidation of cells (particularly, in the membrane of cells), and a reagent for use in the method. More specifically, the present invention provides
The present invention relates to a method for analyzing peroxidation of cells (particularly, in the membrane of cells), and a reagent for analyzing peroxidation of cells, wherein diphenyl-1-pyrenylphosphine (DPPP) is used as a fluorescent probe. .

[0002]

2. Description of the Related Art Lipid peroxidation has been implicated in the pathogenesis of various diseases including atherosclerosis (Halliwel).
l, B. and Gutteridge, JMC (1999) Free Radicals
in Biology and Medicine.3 rd edn., Oxford Universi
ty Press, Oxford). Polyunsaturated fatty acids and their esters are major targets for reactive oxygen species and reactive nitrogen species, and their oxidation in membranes is thought to play an important role in oxidative stress in vivo.
The degree of lipid peroxidation is determined by the thiobarbituric acid reactive substance (Yagi, K. (1984) Methods Enzymol. 105.328-33).
1), lipid hydroperoxide itself (Yamamoto. Y., Bro
dsky. MH, Baker. JC and Ames. BN (1987) Ana
l. Biochem. 160.7-13), and their degradation products such as malondialdehyde and 4-hydroxy-2-nonenal (Bailey, AL, Wortley. G. and Southon. S.
(1997) Free Radical Biol. Med. 23.1078-1085).

[0003] More recently, monoclonal antibodies to 4-hydroxy-2-nonenal and acrolein have also been produced and used (Uchida. K., Osawa, T., Hiai.
H. and Toyokuni. S. (1995) Biochem. Biophys. Res.
Commun. 212, 1068-1073; and Uchida. K., Kanemats
u, M., Sakai, K., Matsuda. T., Hattori. N., Mizun
o, Y., Suzuki, D., Miyata. T., Noguchi, N., Niki.
E. and Osawa. T. (1998) Proc. Natl. Acad. Sci. USA
95. 4882-4887). Each of these methods has advantages and disadvantages.

[0004] To follow the oxidation that occurs in cells and tissues, dichlorodihydrofluorescein diacetate (DCFH-DA) (Tsuchiya. M., Suematsu. M. and
Suzuki. H. (1994) Methods Enzymol. 233.128-140) and dihydrorhodamine (DHR) 123 (Rothe. G. an
d Valet. G. (1994) Methods Enzymol. 233.539-548)
Such fluorescent dyes are widely used. DCFH-DA
Is trapped intracellularly in the form of DCFH. DCFH
And DHR123 itself shows no fluorescence, and generates a when oxidized fluorescent compound H ₂ O ₂ and other oxidant. Whether H ₂ O ₂ is directly reacting with DCFH has been debated (LeBel, CP, Ischiropoulos, H. and
Bondy, SC (1992) Chem. Reg. Toxicol. 5.227-23
1; and Rota. C., Chignell. CF and Mason. RP (1
999) Free Radical Biol. Med. 27.873-881). Fluorescence analysis allows continuous observation of living cells and tissues under a microscope during oxidation. However, since lipid peroxidation occurs in the membrane, the hydrophilic compounds DCFH and DHR123 are unsuitable for measuring lipid peroxidation.

Diphenyl-1-pyrenylphosphine (D
(PPP) is known to react stoichiometrically with peroxide to form diphenyl-1-pyrenylphosphine oxide (DPPP = O) and an alcohol (water if the peroxide is hydrogen peroxide). (See equation below) (Ak
asaka, K., Suzuki, T., Ourui.H. and Meguro.H. (19
87) Anal. Lett. 20.731-745, 797-807).

[0006]

Embedded image

Diphenyl-1-pyrenylphosphine (D
(PPP) is a non-fluorescent molecule, while diphenyl-1-pyrenylphosphine oxide (DPPP = O) is a fluorescent molecule. Plasma levels of phosphatidylcholine, phosphatidylethanolamine, triglycerol and cholesteryl ester hydroperoxides are
It is measured by a high-performance liquid chromatography (HPLC) post-column detection system using 1-pyrenylphosphine (DPPP) (Akasaka. K., Ohrui. H. a.
nd Meguro. H., (1993) J. Chromatogr. 617.205-211;
Akasaka. K., Ohta. A., Ohrui. H. and Megro. H. (19
95) J. Chromatogr. 622.153-159; and Akasaka. K., Oh
ta. A., Ohrui. H. and Meguro. H. (1995) J. Chromat
ogr. B 665. 37-43). However, methods for analyzing the peroxidation status of lipids in living cells have so far been:
Not reported.

[0008]

An object of the present invention is to provide a method for analyzing peroxidation of cells, particularly a method for selectively analyzing peroxidation of lipids in cell membranes of cells. Another object of the present invention is to provide a method for analyzing peroxidation of cells without destroying the cells. Another object of the present invention is to provide a method for easily and visually analyzing the peroxidation state of cells over time. Another object of the present invention is to provide a reagent for analyzing cell peroxidation.

[0009]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, have found that diphenyl-
1-pyrenylphosphine (DPPP) was administered and diphenyl-1-pyrenylphosphine oxide (DPPP =
It has been found that the peroxidation of cells can be analyzed by measuring the fluorescence of O), and the present invention has been completed.

That is, according to the present invention, diphenyl-1-pyrenylphosphine (DPPP) is administered to cells, and diphenyl-1-pyrenylphosphine oxide (DPPP =
There is provided a method for analyzing peroxidation of cells, which comprises measuring the fluorescence of O). The cells are preferably animal cells, more preferably phagocytes, particularly preferably neutrophils or macrophages. In the present invention, preferably, the fluorescence of diphenyl-1-pyrenylphosphine oxide (DPPP = O) is measured using a fluorescence microscope.

In the present invention, preferably, cell membrane peroxidation is analyzed, and specifically, lipids and / or
Alternatively, protein peroxidation is analyzed. In the present invention, preferably, diphenyl-1-pyrenylphosphine (DPPP) incorporated into a lipoprotein is administered to a cell, and the peroxidation state of the lipoprotein in the cell is monitored. Preferably, in the present invention, peroxidation is analyzed without destroying the cells. According to another aspect of the present invention, there is provided a reagent comprising diphenyl-1-pyrenylphosphine (DPPP) for analyzing peroxidation of cells, especially living cells.

[0012]

Embodiments of the present invention will be described below in detail. In the method for analyzing peroxidation of cells according to the present invention, diphenyl-1-pyrenylphosphine (DPPP) is administered to cells to be analyzed, and the fluorescence of diphenyl-1-pyrenylphosphine oxide (DPPP = O) is measured. It is characterized by measuring. Diphenyl-1-pyrenylphosphine (DPPP) is a non-fluorescent substance, but diphenyl-1-pyrenylphosphine oxide (DPPP = O) generated by reaction with peroxide is a fluorescent substance. By measuring the fluorescence of -1-pyrenylphosphine oxide (DPPP = O), the presence and amount of peroxide in cells can be measured.

The type of cells to which the analysis method of the present invention can be applied is not particularly limited, but is generally an animal cell.
Cells to be subjected to the analysis can be appropriately selected depending on the purpose of the analysis. As the cells, for example, phagocytes can be used, and specifically, polymorphonuclear leukocytes (neutrophils, eosinophils, basophils, etc.) and mononuclear phagocytes (monocytes, macrophages, etc.) Is mentioned. Particularly preferred cells for use in the present invention are neutrophils and macrophages.

In the analysis method of the present invention, diphenyl-1-
The fluorescence of pyrenylphosphine oxide (DPPP = O) is measured using a fluorescence microscope. In the present invention, diphenyl-1-pyrenylphosphine oxide (DPPP =
Excitation and fluorescence wavelengths for detecting O) include 351 nm and 380 nm, respectively. The fluorescence can be qualitatively or quantitatively detected by measuring the fluorescence energy generated by irradiating a certain excitation light. At the time of quantification, the intensity of the fluorescent energy can be evaluated as an index of the abundance of the subject. The fluorescence energy may be measured using a suitable commercially available detector.

In one embodiment of the present invention, the membranes of cells are analyzed for peroxidation, and more specifically, membrane lipids and / or proteins are analyzed for peroxidation. Lipids are more easily oxidized than nucleic acids and proteins, and lipid hydroperoxides (LO
OH) and further decomposed aldehyde compounds, which are further reacted with the protein to form a complex modified form, resulting in degradation of the protein. Hydroperoxides generated at the beginning of lipid peroxidation have been used as oxidative stress markers. Lipid peroxidation is a reaction in which oxygen is added to the unsaturated fatty acid portion of the lipid that is a component of the cell membrane. As a pathological condition, macrophages take in LDL in which the lipid portion has been oxidized and are converted into foam cells. Curing. In the field of clinical testing, it can be used for estimating in vivo production of active oxygen, oxygen damage to organs, deterioration of defense mechanisms, and the like. In the analysis method of the present invention, the presence of a hydroperoxide of a cell membrane lipid can be detected and measured.

In one embodiment of the present invention, diphenyl-1-pyrenylphosphine (DPPP) incorporated into a lipoprotein is administered to a cell, and the peroxidation state of the lipoprotein in the cell is monitored. By observing the intracellular fluorescence with a fluorescence microscope over time, the state of peroxidation in the cell can be tracked. The method of the present invention can analyze peroxidation of a cell without destroying the cell and keeping the cell alive. Therefore, it is particularly useful for analysis of a disease state model of a disease involving peroxidation of cell membrane lipid such as arteriosclerosis.

The method for analyzing peroxidation of cells of the present invention has the following advantages. (1) Diphenyl-1-pyrenylphosphine (DPP
P) reacts with the peroxide stoichiometrically (molar to molar ratio) to form diphenyl-1-pyrenylphosphine oxide (DP)
PP = O), which is suitable for quantitatively measuring lipid peroxide. This is an advantage over the chemiluminescence method. This is because the chemiluminescence method has high sensitivity but is not necessarily suitable for quantitative analysis. (2) Diphenyl-1-pyrenylphosphine (DPP
Because P) is very lipophilic, it reacts selectively with lipid peroxides and does not react with peroxides in aqueous solutions. This is an advantage not found in the known fluorescent substances DCFH and DHR123. Diphenyl-1-pyrenylphosphine (DPP
By utilizing P), it is possible to selectively analyze only lipid peroxidation generated in the membrane. (3) Diphenyl-1-pyrenylphosphine (DPP
The analysis method of the present invention using P) has high sensitivity and 1 fmo.
It is possible to detect l peroxide.

The present invention further relates to a reagent for analyzing peroxidation of cells, comprising diphenyl-1-pyrenylphosphine (DPPP). Diphenyl-1-pyrenylphosphine (DPPP) itself used in the reagent of the present invention is a known compound and can be easily obtained from commercial products and the like.
In the present invention, diphenyl-1-pyrenylphosphine (DP
PP) is used to analyze cellular peroxidation. The form of the reagent containing diphenyl-1-pyrenylphosphine (DPPP) is not particularly limited, and may be a solid or a liquid (solution, suspension, etc.). In the case of a liquid, a suitable solvent (preferably diphenyl-1-pyrenylphosphine (DPPP))
The reagent can be prepared by dissolving or suspending the compound in an organic solvent having a certain solubility. The reagent of the present invention is for use in the method for analyzing peroxidation of a cell according to the present invention, and the type of cell and the mode of analyzing peroxidation herein are the same as those described herein above. The same is true. The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to the examples.

[0019]

EXAMPLES Example 1: Follow-up experiment of neutrophil oxidation (1) Materials and methods Reagents dioleoylphosphatidylcholine (DOPC), te
rt-butyl hydroperoxide (t-BuOOH) and phorbol 12-myristate 13-acetate (PM
A) was obtained from Sigma (St. Louis, MO, USA). Hydrogen peroxide (H ₂ O ₂₎ was purchased from Wako Pure Chemical Industries (Osaka, Japan). Diphenyl-1-pyrenylphosphine (hereinafter referred to as DP
PP) was obtained from Dojin Chemical (Kumamoto, Japan). Methyl linoleate hydroperoxide (MeLOO
H) HPLC from methyl linoleate obtained from Sigma
And purified as previously reported (Yamamoto. Y., Niki.
E., Kamiya. Y. and Shimasaki, H. (1984) Biochem. B
iophys. Acta 795, 332-340; and Noguchi. N., Numano.
R., Kaneda.H. And Niki.E. (1998) Free Radical R
es. 29. 43-52), and quantified by reaction with triphenylphosphine.

Oxidation procedure in a cell-free system An ethanol solution (500 μl) containing 20 mM DOPC and 1 mM DPPP was stirred into 10 ml of phosphate buffered saline (PBS, 10 mM, pH 7.4) while stirring.
Monolamellar vesicles were prepared by injection at 7 ° C. The solution was further diluted with PBS and the final concentrations of DOPC and DPPP were adjusted to 20 μM and 1 μM, respectively. H ₂ O ₂ ,
The reactivity of MePPOH and t-BuOOH with DPPP was evaluated at 37 ° C. in chloroform / methanol (1/1) and liposome suspensions containing 10 μM peroxide and 1 μM DPPP. Oxidation of DPPP is achieved by using a thermostatically controlled cuvette with Pe.
Fluorescence was followed every 30 seconds for 5 minutes using an rkin Elmer LB50B fluorescence spectrophotometer. Excitation and emission wavelengths respectively
351 nm (slit width 5.0 nm) and 380 nm (slit width 5.0 nm). Solutions of DPPP without peroxide and liposome suspensions were used as negative controls.

DPPP uptake into mouse neutrophils (PMN)
Write mouse PMN is female 23~28g ICR (Charles Rive
r) with 2 ml of 4% thioglycolate (Difco Laboratorie
s) Five hours after the solution was injected intraperitoneally (ip), the solution was collected from the peritoneal cavity with PBS. Cells were washed three times with PBS and adjusted to 1 × 10 ⁷ cells / ml. After adjusting the 5 mM DMSO solution,
The cells were added at a final concentration of 50 μM and incubated at 37 ° C. for 10 minutes. Then the cells twice, Hanks buffer containing Ca ²⁺ and Mg ²⁺ (Hanks' balanced salt soluti
on; HBSS).

Procedure for Oxidation of PMN Various concentrations of H ₂ O ₂ and methyl linoleate hydroperoxide (MeLOOH) were added to PMN (5
× 10 ⁵ cells / ml). After incubation at 37 ° C. for 60 minutes, the fluorescence intensity of the cell suspension was measured using a fluorescence spectrophotometer. Excitation and emission wavelengths of 35 each
1 nm (slit width 5.0 nm) and 380 nm (slit width 5.0 nm). The oxidation of PMN when stimulated with PMA (Sigma) was also measured. 1 PMN containing DPPP (5 × 10 ⁵ / ml)
It was activated with 00nM PMA to induce oxidation of PMN itself, and the fluorescence intensity was followed for 4 hours at 37 ° C.

[Image analysis] Image analysis is 100W mercury lamp and monochrome CCD camera
This was performed using an Axiovert fluorescence microscope (Zeiss) equipped with (Sony). The lens is a 40x oil immersion lens (Olympus, 1.3N.
A.,) and the bandpass filter is 351 nm (slit width
16 nm), the dichroic mirror is 365 nm, the barrier filter is 380 nm (slit width 20 nm), and diphenyl-1-
It was set so that the fluorescence of pyrenyl phosphine oxide (hereinafter abbreviated as DPPP = O) selectively passed. C
Images from the CD camera were accumulated for one second. At the time of image analysis, PMN (1 × 10 ⁶ / ml) was converted to HBSS (1% fatal bovine).
serum; FBS) and attached to a 35 nm glass base dish (Iwaki). DPPP was added at a final concentration of 5 μM, incubated for 15 minutes and taken up by cells. Thereafter, washing with HBSS (1% FBS) was performed to remove excess DPPP not taken up by cells. After placing the sample on a microscope, 5 μM MeLOOH was added as a methanol solution, and the fluorescence of DPPP = O was photographed in the same visual field for 30 minutes.

(2) Results DPPP and peroxidation in organic solution and liposome membrane
Reaction with Matter DPPP is known to react with peroxide to produce fluorescent DPPP = O (Akasaka, K., Suzuki,
T., Ourui. H. and Meguro. H. (1987) Anal. Lett. 20.
731-745, 797-807). First, the reactivity between DPPP and peroxide in a homogeneous solution was examined. As shown in FIG.
When M peroxide was reacted with 1 μM DPPP in chloroform / methanol (1/1) at 37 ° C., the fluorescence intensity measured by fluorescence spectrophotometry increased linearly for 5 minutes. DPPP = O is 10 μM each of MeLOO
When reacted with H, H ₂ O ₂ , and tBuOOH, they formed 3.8, 3.3, and 1.2 times more than controls without peroxide. MeLOOH and H ₂ O ₂
It showed higher reactivity to DPPP as compared to tBuOOH.

A liposome membrane is often used as a model for a biofilm.
used. DPP incorporated into DOPC liposome membrane
Reacts P with peroxide and fluoresces increase in DPPP = O
Followed by spectrophotometry. The results are shown in FIG. 1B. Excessive
The oxide was added as a methanol solution. MeLOOH
Is added to the liposome suspension to form DPPP = O
The growth reaches the plateau in 1 minute, and the fluorescence level is controlled.
Was 22 times higher. H _TwoO_TwoAnd by t-BuOOH
The increase in DPPP = O was slight. DPPP is uniform
In both solution and liposome solution
Oxidized spontaneously.

Formation of DPPP = O in PMN First, the uptake and stability of DPPP = O in PMN were examined. DPPP = O was incorporated into the cells and extracted with chloroform / methanol (2/1). When the amount of DPPP = O taken into the cells was calculated by oxidizing the whole amount, it was 0.35 nmol / 10 ⁶ cells, and DPPP = O
Was stable in PMN for at least 4 hours. P
Incorporate DPPP into MN, H ₂ O ₂ and MeLOO
Reaction with H revealed that DPPP in PMN was more efficiently oxidized by MeLOOH than H ₂ O ₂ , as observed on the liposome membrane (FIG. 2). This indicates that DPPP selectively reacts with lipid peroxide in cells.

PMN containing DPPP (1 × 10 ⁶ pieces / m
When l) was stimulated with PMA, DPPP = O was generated and the fluorescence increased over time (FIG. 3). The production of DPPP = 0 rarely occurred without stimulation. This demonstrates that the intracellular oxidation reaction of PMN when stimulated with PMA can be traced by the generation of DPPP = O fluorescence. Observation of DPPP = O with a microscope was also attempted. Thirty minutes after adding 5 μM MeLOOH to PMN, the increased fluorescence could be visualized by microscopy (FIGS. 4A, B). When no peroxide is added, the fluorescence hardly increases (FIG. 4C). DPPP =
O was observed to be distributed in the cells, but the nucleus was not stained. Almost no fluorescence was observed in cells that did not incorporate DPPP.

Example 2: Follow-up experiment of oxidation of lipoprotein in cells (1) Method DPPP uptake into LDL Low-density lipoprotein (LDL) was separated from frozen human plasma by ultracentrifugation. 10% Pluronic F-127 (Molecul
ar Probe) was added to a DMSO solution of DPPP to a concentration of 2.5 mM. This DPPP solution was added to 2 mg (protein) / ml (PBS) of LDL to a concentration of 2%, and incubated at 37 ° C. for 20 minutes to reduce DPPP to L.
DL was taken in. Then, LDL was purified again by ultracentrifugation, and the amount of protein and DPP incorporated in LDL
The amount of P was calculated. The amount of DPPP incorporated into LDL is determined by the fluorescence intensity of a 0.1 μM LDL solution and
DL was reacted with 10 μM MeLOOH at 37 ° C. for 40 minutes to completely oxidize DPPP, and the fluorescence intensity was measured and calculated. According to it, about 10 DPPs per LDL particle
It was found that P was incorporated, and 2-3 of them had already been oxidized. Similarly, instead of DPPP, DPPP =
L was labeled with O. The incorporation of both into LDL was comparable.

[0029] Macrophage collection of DPPP-labeled LDL
Write mouse macrophages female 23-28g ICR (Charles Rive
r) 2 ml of 4% thioglycolate (Difco Laboratories)
The solution was collected from the peritoneal cavity with PBS 72 hours after intraperitoneal (ip) administration. The cells were washed three times with PBS, adjusted to 5 × 10 ⁵ cells / ml, and then attached to a culture dish. 0.1 μM of DPPP-labeled LDL and DPPP = O-labeled LDL were added thereto, and the cells were cultured at 37 ° C. for 0, 2, and 4 hours.

(2) Results LDL incorporation into macrophages and fluorescence of DPPP = O in macrophages 0, 2, and 4 hours after oxidation, were observed with a fluorescence microscope.
A comparison was made using PP = O-labeled LDL (FIG. 5).
When DPPP = O-labeled LDL was added, the fluorescence increased in a time-dependent manner, and it was confirmed that LDL was incorporated into the cells. When DPPP-labeled LDL was added, the fluorescence increased in a time-dependent manner, but the fluorescence after 0 and 2 hours was less than when DPPP = O-labeled LDL was added. On the other hand, the fluorescence after 4 hours was almost the same as when DPPP = O-labeled LDL was added. This suggests that DPPP-labeled LDL may be oxidized in the cells over a period of 2 to 4 hours, assuming that the rate of incorporation into both cells is approximately the same.

[0031]

According to the present invention, a new method for analyzing cell peroxidation, particularly a new method for selectively analyzing cell membrane lipid peroxidation, is provided. According to the method of the present invention, peroxidation of the membrane of a living cell can be directly detected as a fluorescent signal. Therefore, by utilizing the method of the present invention, it is possible to easily and visually analyze the peroxidation over time without breaking the cells. The method for analyzing peroxidation of cells according to the present invention is useful for studies such as elucidation of the mechanism and significance of oxidative degeneration in the onset of arteriosclerosis and elucidation of the relationship between aging and oxidative stress in aging model animals such as nematodes.

[Brief description of the drawings]

FIG. 1 shows chloroform / methanol (1/1)
(FIG. 1A) and DOPC liposome membrane (FIG. 1B)
4 is a graph showing DPPP oxidation by peroxides in FIG. MeLOOH, was followed by fluorescence spectrophotometry measuring the formation of DPPP = O in the absence (none) or presence of H ₂ O ₂ or peroxides, such as tBuOOH,. All experiments were performed at 37 ° C. in air, with initial concentrations of DPPP and peroxide of 1 μM and 10 μM, respectively. Values are means ± S.D. D. (N = 3).

FIG. 2 shows PM with H ₂ O ₂ and MeLOOH.
3 shows the oxidation of DPPP in N. Mouse PMN (1
× 10 ⁷ cells / ml) were incubated with 50 μM DPPP at 37 ° C. for 10 minutes, washed, and adjusted to 5 × 10 ⁵ cells / ml. The indicated concentrations of H ₂ O ₂ and MeLOOH were added and 3
Incubated at 7 ° C for 60 minutes. DPPP generated
= O was determined from the increase in fluorescence intensity of the cell suspension and was expressed as mean ± SEM. D. (N = 3).

FIG. 3 shows lipid peroxide formation in PMA stimulated PMN. Mouse PMN (1 × 10 ⁷ cells / ml) was incubated with 50 μM DPPP at 37 ° C. for 10 minutes, washed, and adjusted to 5 × 10 ⁵ cells / ml. 10 PMN
Stimulated with 0 nM PMA and incubated for 4 hours at 37 ° C. The amount of DPPP = O produced was calculated from the increase in the fluorescence intensity of the cell suspension, and the mean ± S.D. D. (N = 3).

FIG. 4 shows visualization of lipid oxidation in PMN.
PMN (1 × 10 ⁶ cells / ml) was stained with 5 μM DPPP for 15 minutes, and washed with HBSS containing 1% FBS. After addition of 5 μM MeLOOH (A: 0 min and B: 30 min)
It shows an increase in fluorescence without the addition of H ₂ O ₂ (C: 30 minutes).

[FIG. 5] FIG. 5 is a graph showing that DPPP-labeled LDL and DPPP = O-labeled LDL have been incorporated into macrophages.
The result of observing the fluorescence of DPPP = O in macrophages with a fluorescence microscope is shown.

 ──────────────────────────────────────────────────続 き Continued on the front page (71) Applicant 500474996 Tatsuhiko Kodama 4-16-5 Shimoma, Setagaya-ku, Tokyo (71) Applicant 500475007 Yoichiro Wada 2-10-45-503, Mita, Meguro-ku, Tokyo (71) Applicant 500475856 Ryo Watanabe 403-6 YM Building 1-23-6 Mejirodai, Bunkyo-ku, Tokyo (71) Applicant 500475018 Shun Yamashita 860-4 Higashifukai, Nagareyama-shi, Chiba (72) Inventor Noriko Noguchi 2-9-12 Hanegi, Setagaya-ku, Tokyo (72) Inventor Yuko Okimoto 4-3-41-103 Meguro Sanda Park Mansion, Komaba, Meguro-ku, Tokyo (72) Inventor Tetsuo Futaki 7-1-2, Yoto, Utsunomiya City, Tochigi Prefecture (72) Inventor Tatsuhiko Kodama Tokyo 4-16-5 Shimouma, Setagaya-ku, Tokyo (72) Inventor Yoichiro Wada 2-10-45-503, Mita, Meguro-ku, Tokyo (72) Inventor Ryo Watanabe 1-23-6 YM Building, Mejirodai, Bunkyo-ku, Tokyo 403 (72) Inventor Shun Yamashita 860-4 Higashi Fukai, Nagareyama-shi, Chiba F-term (reference) AB02 CA07 CE02 EA03 GA04 4B063 QA01 QA18 QQ08 QQ70 QR41 QS03 QS36 QX02

Claims (11)

[Claims] 1. A method for peroxidizing cells, comprising administering diphenyl-1-pyrenylphosphine (DPPP) to cells and measuring the fluorescence of diphenyl-1-pyrenylphosphine oxide (DPPP = O). How to analyze. 2. The method according to claim 1, wherein the cell is an animal cell. 3. The method according to claim 1, wherein the cells are phagocytes. 4. The method according to claim 1, wherein the cells are neutrophils or macrophages. 5. The method according to claim 1, wherein the fluorescence of diphenyl-1-pyrenylphosphine oxide (DPPP) is measured using a fluorescence microscope. 6. The method of claim 1, wherein the cell membrane is analyzed for peroxidation.
6. The method according to any one of claims 1 to 5. 7. The method according to claim 1, wherein cell membrane lipids and / or proteins are analyzed for peroxidation. 8. The method according to claim 1, wherein diphenyl-1-pyrenylphosphine (DPPP) incorporated in the lipoprotein is administered to the cell, and the peroxidation state of the lipoprotein in the cell is tracked. The method according to any one of the above. 9. The analysis method according to claim 1, wherein peroxidation is analyzed without destroying cells. 10. A reagent for analyzing cellular peroxidation, comprising diphenyl-1-pyrenylphosphine (DPPP). 11. The reagent according to claim 10, for analyzing live cells for peroxidation.