JP2006126073A - Method of detecting membrane potential change, method of screening drug and well plate - Google Patents
Method of detecting membrane potential change, method of screening drug and well plate Download PDFInfo
- Publication number
- JP2006126073A JP2006126073A JP2004316512A JP2004316512A JP2006126073A JP 2006126073 A JP2006126073 A JP 2006126073A JP 2004316512 A JP2004316512 A JP 2004316512A JP 2004316512 A JP2004316512 A JP 2004316512A JP 2006126073 A JP2006126073 A JP 2006126073A
- Authority
- JP
- Japan
- Prior art keywords
- dye
- cell
- membrane potential
- fluorescence
- membrane
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 239000012528 membrane Substances 0.000 title claims abstract description 121
- 230000008859 change Effects 0.000 title claims abstract description 72
- 238000000034 method Methods 0.000 title claims abstract description 37
- 229940079593 drug Drugs 0.000 title claims description 27
- 239000003814 drug Substances 0.000 title claims description 27
- 238000012216 screening Methods 0.000 title claims description 14
- 210000004027 cell Anatomy 0.000 claims abstract description 102
- 238000010791 quenching Methods 0.000 claims abstract description 46
- 230000000171 quenching effect Effects 0.000 claims abstract description 46
- 210000000170 cell membrane Anatomy 0.000 claims abstract description 37
- 238000010186 staining Methods 0.000 claims abstract description 21
- 230000003834 intracellular effect Effects 0.000 claims abstract description 17
- 230000005284 excitation Effects 0.000 claims abstract description 14
- 230000001678 irradiating effect Effects 0.000 claims abstract description 6
- 239000007850 fluorescent dye Substances 0.000 claims description 63
- 239000000975 dye Substances 0.000 claims description 58
- 238000005259 measurement Methods 0.000 claims description 34
- 102000004310 Ion Channels Human genes 0.000 claims description 26
- 108091006671 Ion Transporter Proteins 0.000 claims description 24
- 102000037862 Ion Transporter Human genes 0.000 claims description 24
- 239000000049 pigment Substances 0.000 abstract 4
- 238000001514 detection method Methods 0.000 description 26
- 239000002609 medium Substances 0.000 description 22
- 239000000243 solution Substances 0.000 description 13
- 238000007877 drug screening Methods 0.000 description 11
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 10
- 210000004509 vascular smooth muscle cell Anatomy 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 6
- 230000004044 response Effects 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- 230000035945 sensitivity Effects 0.000 description 5
- 239000012192 staining solution Substances 0.000 description 5
- 235000009328 Amaranthus caudatus Nutrition 0.000 description 4
- 240000001592 Amaranthus caudatus Species 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 239000004178 amaranth Substances 0.000 description 4
- 235000012735 amaranth Nutrition 0.000 description 4
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- 239000000556 agonist Substances 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 230000007423 decrease Effects 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- 238000013537 high throughput screening Methods 0.000 description 3
- 239000003068 molecular probe Substances 0.000 description 3
- 230000003204 osmotic effect Effects 0.000 description 3
- 235000013824 polyphenols Nutrition 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- -1 acetoxymethyl group Chemical group 0.000 description 2
- ZYGHJZDHTFUPRJ-UHFFFAOYSA-N coumarin Chemical compound C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 210000000805 cytoplasm Anatomy 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 230000032050 esterification Effects 0.000 description 2
- 238000005886 esterification reaction Methods 0.000 description 2
- 229930003935 flavonoid Natural products 0.000 description 2
- 150000002215 flavonoids Chemical class 0.000 description 2
- 235000017173 flavonoids Nutrition 0.000 description 2
- 238000001000 micrograph Methods 0.000 description 2
- 230000035699 permeability Effects 0.000 description 2
- 230000008884 pinocytosis Effects 0.000 description 2
- 150000008442 polyphenolic compounds Chemical class 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 230000002123 temporal effect Effects 0.000 description 2
- 230000036962 time dependent Effects 0.000 description 2
- 229910052724 xenon Inorganic materials 0.000 description 2
- FHNFHKCVQCLJFQ-UHFFFAOYSA-N xenon atom Chemical compound [Xe] FHNFHKCVQCLJFQ-UHFFFAOYSA-N 0.000 description 2
- KUINQLKLKSFJME-UFRBDIPQSA-N (4z)-4-[(2e,4e)-5-(5-oxo-3-phenyl-2h-1,2-oxazol-4-yl)penta-2,4-dienylidene]-3-phenyl-1,2-oxazol-5-one Chemical compound O=C1ON=C(C=2C=CC=CC=2)\C1=C\C=C\C=C\C(C(ON1)=O)=C1C1=CC=CC=C1 KUINQLKLKSFJME-UFRBDIPQSA-N 0.000 description 1
- HAPJROQJVSPKCJ-UHFFFAOYSA-N 3-[4-[2-[6-(dibutylamino)naphthalen-2-yl]ethenyl]pyridin-1-ium-1-yl]propane-1-sulfonate Chemical compound C1=CC2=CC(N(CCCC)CCCC)=CC=C2C=C1C=CC1=CC=[N+](CCCS([O-])(=O)=O)C=C1 HAPJROQJVSPKCJ-UHFFFAOYSA-N 0.000 description 1
- IXFSUSNUALIXLU-UHFFFAOYSA-N 3-[4-[2-[6-(dioctylamino)naphthalen-2-yl]ethenyl]pyridin-1-ium-1-yl]propane-1-sulfonate Chemical compound C1=CC2=CC(N(CCCCCCCC)CCCCCCCC)=CC=C2C=C1C=CC1=CC=[N+](CCCS([O-])(=O)=O)C=C1 IXFSUSNUALIXLU-UHFFFAOYSA-N 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 229940116531 Ion channel agonist Drugs 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 235000010208 anthocyanin Nutrition 0.000 description 1
- 239000004410 anthocyanin Substances 0.000 description 1
- 229930002877 anthocyanin Natural products 0.000 description 1
- 150000004636 anthocyanins Chemical class 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 229910052786 argon Inorganic materials 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- HQMRIBYCTLBDAK-UHFFFAOYSA-M bis(2-methylpropyl)alumanylium;chloride Chemical compound CC(C)C[Al](Cl)CC(C)C HQMRIBYCTLBDAK-UHFFFAOYSA-M 0.000 description 1
- 239000001045 blue dye Substances 0.000 description 1
- 239000000298 carbocyanine Substances 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- IQFVPQOLBLOTPF-HKXUKFGYSA-L congo red Chemical compound [Na+].[Na+].C1=CC=CC2=C(N)C(/N=N/C3=CC=C(C=C3)C3=CC=C(C=C3)/N=N/C3=C(C4=CC=CC=C4C(=C3)S([O-])(=O)=O)N)=CC(S([O-])(=O)=O)=C21 IQFVPQOLBLOTPF-HKXUKFGYSA-L 0.000 description 1
- 229960000956 coumarin Drugs 0.000 description 1
- 235000001671 coumarin Nutrition 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 238000002073 fluorescence micrograph Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000002102 hyperpolarization Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 210000004020 intracellular membrane Anatomy 0.000 description 1
- 230000037427 ion transport Effects 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 230000028161 membrane depolarization Effects 0.000 description 1
- DZVCFNFOPIZQKX-LTHRDKTGSA-M merocyanine Chemical compound [Na+].O=C1N(CCCC)C(=O)N(CCCC)C(=O)C1=C\C=C\C=C/1N(CCCS([O-])(=O)=O)C2=CC=CC=C2O\1 DZVCFNFOPIZQKX-LTHRDKTGSA-M 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 230000003114 pinocytic effect Effects 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000001044 red dye Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
Landscapes
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
本発明は、膜電位変化検出方法、薬物スクリーニング方法、及び膜電位変化検出又は薬物スクリーニングのためのウェルプレートに関する。 The present invention relates to a membrane potential change detection method, a drug screening method, and a well plate for membrane potential change detection or drug screening.
近年、膜電位（細胞膜の内側・外側間の電位差）の変化を検出する手法として膜電位感受性蛍光色素の利用が注目され、実際に、抗生物質の効力の検定やイオンチャネル開口薬の探索に利用されるようになっている（非特許文献１）。
膜電位感受性蛍光色素は、膜電位の変化に応じて細胞膜外側表面及び細胞内への分布の割合が変化する蛍光物質であり、膜電位が上昇する（脱分極に向かう）と細胞内への分布が増加し、逆に膜電位が下降する（過分極に向かう）と細胞膜外側表面への分布が増加するという性質を有する。 A membrane potential-sensitive fluorescent dye is a fluorescent substance whose distribution ratio changes to the outer surface of the cell membrane and into the cell in accordance with changes in the membrane potential. When the membrane potential increases (towards depolarization), the distribution into the cell On the contrary, when the membrane potential decreases (towards hyperpolarization), the distribution to the outer surface of the cell membrane increases.
このような膜電位感受性蛍光色素を利用すれば、この色素から生じる蛍光強度を経時的に測定することによって、膜電位の変化を検出することができる。細胞質中で細胞内タンパク質や細胞内膜に結合することにより蛍光が増強する膜電位感受性蛍光色素（DiBAC4(3)など）を用いる場合には、通常の蛍光顕微鏡で細胞全体の蛍光色素に由来する蛍光強度を経時的に測定すればよいが、その他の膜電位感受性蛍光色素を用いる場合には、細胞膜の内側又は外側の蛍光色素に由来する蛍光をコンフォーカル顕微鏡により検出し、その強度を測定する必要がある。 If such a membrane potential sensitive fluorescent dye is used, a change in the membrane potential can be detected by measuring the fluorescence intensity generated from the dye over time. When using membrane-potential sensitive fluorescent dyes (such as DiBAC4 (3)) that enhance fluorescence by binding to intracellular proteins and inner membranes in the cytoplasm, they are derived from whole cell fluorescent dyes using a normal fluorescence microscope. The fluorescence intensity may be measured over time, but when using other membrane potential sensitive fluorescent dyes, the fluorescence derived from the fluorescent dye inside or outside the cell membrane is detected with a confocal microscope and the intensity is measured. There is a need.
いずれの場合にも、測定される蛍光強度の変化量は大きくないため、例えば、イオンチャネル又はイオン輸送体に作用する薬物のハイスループットスクリーニングにおいて利用するには、必ずしも膜電位変化の検出感度が十分ではないという問題がある。この問題は、特に活性化又は不活性化の速度が速い電位依存性イオンチャネル作用薬を探索する場合に顕著である（DOJIN News No.103(2002)）。さらに、膜の内側又は外側の蛍光強度を測定する必要がある場合には、操作が煩雑であるという問題もある。 In any case, since the amount of change in the measured fluorescence intensity is not large, for example, the detection sensitivity of changes in membrane potential is not always sufficient for use in high-throughput screening of drugs acting on ion channels or ion transporters. There is a problem that is not. This problem is particularly noticeable when searching for a voltage-gated ion channel agonist with a high rate of activation or inactivation (DOJIN News No. 103 (2002)). Furthermore, when it is necessary to measure the fluorescence intensity inside or outside the membrane, there is also a problem that the operation is complicated.
そこで、本発明は、膜電位感受性蛍光色素を利用した、より高感度で簡便な膜電位変化検出方法を提供することを目的とする。 Accordingly, an object of the present invention is to provide a more sensitive and simple method for detecting a change in membrane potential using a membrane potential sensitive fluorescent dye.
上記目的を達成するために、本発明は、細胞内又は細胞外媒体への蛍光消光色素の導入と、膜電位感受性蛍光色素による前記細胞の細胞膜の染色とを、同時に又はいずれか一方を先に実施して色素導入細胞を得るステップ（以下場合により「導入ステップ」という。）と、前記色素導入細胞に励起光を照射して、前記膜電位感受性蛍光色素から生じる蛍光の強度を経時的に測定するステップ（以下場合により「測定ステップ」という。）と、を備える膜電位変化検出方法を提供する。 In order to achieve the above object, the present invention provides a method of introducing a fluorescence quenching dye into a cell or an extracellular medium and staining the cell membrane of the cell with a membrane potential-sensitive fluorescent dye, either at the same time or first. A step of obtaining dye-introduced cells (hereinafter referred to as “introduction step”), and irradiating the dye-introduced cells with excitation light to measure the intensity of fluorescence generated from the membrane potential-sensitive fluorescent dye over time. And a method for detecting a change in membrane potential (hereinafter, referred to as a “measurement step” in some cases).
蛍光消光色素が細胞内に導入された場合には、細胞内の蛍光色素に由来する蛍光が消光されるので、細胞への励起光の照射により得られる全蛍光の強度は、細胞膜上（外側表面）の蛍光色素に由来する蛍光の強度にほぼ等しくなる。また、蛍光消光色素が細胞外媒体に導入された場合には、細胞外の蛍光色素に由来する蛍光が消光されるので、細胞への励起光の照射により得られる全蛍光の強度は、細胞内の蛍光色素に由来する蛍光の強度にほぼ等しくなる。従って、上記膜電位変化検出方法によれば、膜の内側又は外側の蛍光強度を測定するに当たり、細胞膜上及び細胞内の蛍光色素に由来する全蛍光の強度を測定すれば足り、また、測定される蛍光強度の変化量は膜電位変化をより正確に反映したものとなる。すなわち、膜電位感受性蛍光色素を利用した、より高感度で簡便な膜電位変化検出方法が得られる。 When a fluorescence quenching dye is introduced into a cell, the fluorescence derived from the intracellular fluorescence dye is quenched, so the intensity of the total fluorescence obtained by irradiating the cell with excitation light is on the cell membrane (outer surface ) Of the fluorescent dye derived from the fluorescent dye. In addition, when the fluorescence quenching dye is introduced into the extracellular medium, the fluorescence derived from the extracellular fluorescent dye is quenched, so the intensity of the total fluorescence obtained by irradiating the cell with excitation light is It becomes substantially equal to the intensity of fluorescence derived from the fluorescent dye. Therefore, according to the membrane potential change detection method described above, in measuring the fluorescence intensity inside or outside the membrane, it is sufficient to measure the intensity of the total fluorescence derived from the fluorescent dye on and in the cell membrane. The amount of change in fluorescence intensity more accurately reflects changes in membrane potential. That is, a more sensitive and simple method for detecting a change in membrane potential using a membrane potential sensitive fluorescent dye can be obtained.
本発明の膜電位変化検出方法は、イオンチャネル及び／又はイオン輸送体に作用する薬物のスクリーニング方法として利用することができる。すなわち、本発明は、細胞のイオンチャネル及び／又はイオン輸送体に作用する薬物のスクリーニング方法であって、前記イオンチャネル及び／又はイオン輸送体を前記細胞に発現させるステップ（以下場合により「発現ステップ」という。）と、前記細胞内又は細胞外媒体への蛍光消光色素の導入と、膜電位感受性蛍光色素による前記細胞の細胞膜の染色とを、同時に又はいずれか一方を先に実施して色素導入細胞を得るステップ（以下場合により「導入ステップ」という。）と、候補薬物を前記色素導入細胞に接触させるステップ（以下場合により「接触ステップ」という。）と、前記色素導入細胞に励起光を照射して、前記膜電位感受性蛍光色素から生じる蛍光の強度を経時的に測定することにより、前記候補薬物との接触による前記色素導入細胞の膜電位変化を検出するステップ（以下場合により「検出ステップ」という。）と、を備えるスクリーニング方法を提供する。 The membrane potential change detection method of the present invention can be used as a screening method for drugs that act on ion channels and / or ion transporters. That is, the present invention is a method for screening a drug that acts on an ion channel and / or an ion transporter of a cell, the step of expressing the ion channel and / or the ion transporter in the cell (hereinafter referred to as an “expression step” in some cases). )), Introduction of a fluorescence quenching dye into the cell or extracellular medium, and staining of the cell membrane of the cell with a membrane potential-sensitive fluorescent dye, either at the same time or in advance, the dye introduction. A step of obtaining cells (hereinafter referred to as “introduction step”), a step of bringing a candidate drug into contact with the dye-introduced cells (hereinafter referred to as “contact step”), and irradiation of the dye-introduced cells with excitation light. Then, by measuring the intensity of fluorescence generated from the membrane potential sensitive fluorescent dye over time, the contact with the candidate drug (Optionally hereinafter referred to as "detection step".) Detecting the membrane potential change of the element introduced cells and provides a screening method comprising.
このスクリーニング方法により、膜電位感受性蛍光色素を利用した、より高感度で高効率の、イオンチャネル又はイオン輸送体に作用する薬物のスクリーニング方法が得られる。 This screening method provides a more sensitive and efficient screening method for drugs that act on ion channels or ion transporters using membrane potential sensitive fluorescent dyes.
ここで、イオンチャネルは、電位依存性であるか、リガンド依存性であるかを問わない。イオン輸送体とは、電位発生的なイオン輸送を行う細胞膜の輸送体であり、例として、Ｎａ＋,Ｋ＋−ＡＴＰアーゼ、Ｃａ＋−ＡＴＰアーゼが挙げられる。 Here, it does not matter whether the ion channel is voltage-dependent or ligand-dependent. The ion transporter is a transporter of a cell membrane that performs ionogenic ion transport, and examples thereof include Na + , K + -ATPase, and Ca + -ATPase.
上記膜電位変化検出方法及びスクリーニング方法においては、膜電位変化の検出は、測定開始時の蛍光強度を基準とした蛍光強度の最大変化量、並びに、測定時間−蛍光強度曲線の測定開始時から測定終了時までの積分値を求めて行うのが好ましい。こうすることにより、膜電位変化のより定量的な検出が可能となる。 In the above-described membrane potential change detection method and screening method, the detection of the membrane potential change is measured from the start of measurement of the fluorescence intensity maximum change amount and the measurement time-fluorescence intensity curve based on the fluorescence intensity at the start of measurement. It is preferable to obtain the integral value until the end. By doing so, it is possible to more quantitatively detect changes in membrane potential.
本発明はまた以下のウェルプレートを提供する。（１）細胞内又は細胞外媒体への蛍光消光色素の導入と、膜電位感受性蛍光色素による細胞膜の染色とが行われた細胞を、ウェルプレートのウェル内に備える膜電位変化検出用ウェルプレート、（２）細胞のイオンチャネル及び／又はイオン輸送体に作用する薬物のスクリーニングのためのウェルプレートであって、前記イオンチャネル及び／又はイオン輸送体が発現し、細胞内又は細胞外媒体への蛍光消光色素の導入と、膜電位感受性蛍光色素による細胞膜の染色とが行われた細胞を、ウェルプレートのウェル内に備えるウェルプレート。これらのウェルプレートはそれぞれ、膜電位変化検出用、イオンチャネル及び／又はイオン輸送体に作用する薬物のスクリーニング用、に用いられる。 The present invention also provides the following well plate. (1) A well plate for detecting a change in membrane potential, comprising cells that have been subjected to introduction of a fluorescence quenching dye into cells or extracellular medium and staining of the cell membrane with a membrane potential sensitive fluorescent dye in the well of the well plate, (2) A well plate for screening a drug that acts on an ion channel and / or ion transporter of a cell, wherein the ion channel and / or ion transporter is expressed, and fluorescence to an intracellular or extracellular medium A well plate provided with cells that have been subjected to introduction of a quenching dye and staining of a cell membrane with a membrane potential-sensitive fluorescent dye in the well of the well plate. Each of these well plates is used for detection of changes in membrane potential and for screening for drugs acting on ion channels and / or ion transporters.
本発明により、膜電位感受性蛍光色素を利用した、より高感度で簡便な膜電位変化検出方法が得られる。また、膜電位感受性蛍光色素を利用した、より高感度で高効率の、イオンチャネル又はイオン輸送体に作用する薬物のスクリーニング方法が得られる。 According to the present invention, a more sensitive and simple method for detecting a change in membrane potential using a membrane potential sensitive fluorescent dye can be obtained. In addition, a more sensitive and efficient screening method for drugs that act on ion channels or ion transporters using membrane potential sensitive fluorescent dyes can be obtained.
以下、本発明の好適な実施形態を説明する。 Hereinafter, preferred embodiments of the present invention will be described.
（膜電位変化検出方法）
本発明の膜電位変化検出方法は、上述の導入ステップ及び測定ステップを備えるものである。この方法における導入ステップでは、細胞内又は細胞外媒体への蛍光消光色素の導入を行うが、ここで用いられる蛍光消光色素としては、使用する膜電位感受性蛍光色素の蛍光波長により、例えば、青色色素（エバンスブルー(Sigma, E2129)、ダイレクトブルー(Sigma, D2535)、アシッドブルー（Sigma, A4770）等）、赤色色素（アマランス(Sigma, A1016)、コンゴーレッド(Sigma, C6767)等）、ポリフェノール（フラボノイド系、クマリン系等）が挙げられる。
(Membrane potential change detection method)
The membrane potential change detection method of the present invention includes the introduction step and the measurement step described above. In the introduction step in this method, the fluorescence quenching dye is introduced into the intracellular or extracellular medium. The fluorescence quenching dye used here is, for example, a blue dye depending on the fluorescence wavelength of the membrane potential sensitive fluorescence dye used. (Evans Blue (Sigma, E2129), Direct Blue (Sigma, D2535), Acid Blue (Sigma, A4770), etc.), Red dye (Amaranth (Sigma, A1016), Congo Red (Sigma, C6767), etc.), Polyphenol (flavonoid) System, coumarin system, etc.).
細胞内への蛍光消光色素の導入は、例えば、細胞内の浸透圧を変化させて水分とともにピノサイトーシスにより取り込ませる方法、電気穿孔により細胞膜に孔を開けて取り込ませる方法、マイクロインジェクション法、色素を修飾して細胞内への移行性を高めた後、細胞とともにインキュベートする方法（色素にカルボン酸を導入して、そのカルボキシル基にアセトキシメチル基を結合させてエステル化する方法や、フェノール性水酸基をエステル化する方法）により可能である。色素がタンパク質の場合には、その遺伝子を導入して細胞内で発現させてもよい。 For example, fluorescent quenching dyes can be introduced into cells by changing the osmotic pressure in the cells and incorporating them with water by pinocytosis, or by making pores in the cell membrane by electroporation, microinjection methods, dyes To improve the transferability into the cell by modifying the cell, and then incubating with the cell (introduction of carboxylic acid into the dye and esterification by binding the acetoxymethyl group to the carboxyl group, phenolic hydroxyl group Is possible by the esterification method. When the dye is a protein, the gene may be introduced and expressed in cells.
細胞外媒体とは、細胞周囲で当該細胞に接する媒体（培養液、緩衝液など）のことをいう。細胞外媒体への消光色素の導入は、細胞が存在している媒体に蛍光消光色素を溶解したり、蛍光消光色素が溶解した媒体中に細胞を置いたりすることによって行うことができる。ここで用いられる蛍光消光色素は特に制限されないが、細胞膜透過性の低い色素が好ましく、細胞膜透過性のない色素がさらに好ましい。 The extracellular medium refers to a medium (a culture solution, a buffer solution, etc.) that comes into contact with the cell around the cell. The quenching dye can be introduced into the extracellular medium by dissolving the fluorescence quenching dye in a medium in which cells are present or placing the cells in a medium in which the fluorescence quenching dye is dissolved. The fluorescence quenching dye used here is not particularly limited, but a dye having low cell membrane permeability is preferable, and a dye having no cell membrane permeability is more preferable.
導入ステップにおいては、細胞内又は細胞外媒体への蛍光消光色素の導入と共に、膜電位感受性蛍光色素により細胞の細胞膜の染色を行う。膜電位感受性蛍光色素は、比較的遅い膜電位変化を検出するslow response probeと、比較的早い膜電位変化を検出するfast response probeとに分類され、このいずれを用いてもよいが、本発明の膜電位変化検出方法を薬物のハイスループットスクリーニングにおいて利用する場合には、より膜電位感受性の高いslow response probeを用いるのが好ましい。slow response probeとしては、例えば、Oxonol系色素（OxonolV、OxonolVI、DiSBAC2(3)、DiSBAC4(3)、DiBAC4(3)等）、Carbocyanine系色素（DiSC3(5)等）、Merocyanine系色素が挙げられ、fast response probeとしては、例えば、Styryl系色素（di-8-ANEPPS、di-4-ANEPPS等）が挙げられる。また、ここで用いられる膜電位感受性蛍光色素は、細胞質中で細胞内タンパク質や細胞内膜に結合することにより蛍光が増強する膜電位感受性蛍光色素（DiBAC4(3)など）、及びこれ以外の膜電位感受性蛍光色素のいずれでもよい。 In the introduction step, the cell membrane of the cell is stained with a membrane potential sensitive fluorescent dye together with the introduction of the fluorescence quenching dye into the intracellular or extracellular medium. Membrane potential sensitive fluorescent dyes are classified into a slow response probe that detects a relatively slow membrane potential change and a fast response probe that detects a relatively fast membrane potential change, and either of these may be used. When the membrane potential change detection method is used in high-throughput screening of drugs, it is preferable to use a slow response probe with higher membrane potential sensitivity. Examples of slow response probes include Oxonol dyes (OxonolV, OxonolVI, DiSBAC 2 (3), DiSBAC 4 (3), DiBAC 4 (3), etc.), Carbocyanine dyes (DiSC 3 (5), etc.), and Merocyanine dyes Examples of the fast response probe include dyes such as Stylyl dyes (di-8-ANEPPS, di-4-ANEPPS, etc.). The membrane potential sensitive fluorescent dyes used here are membrane potential sensitive fluorescent dyes (such as DiBAC4 (3)) whose fluorescence is enhanced by binding to intracellular proteins and intracellular membranes in the cytoplasm, and other membranes. Any of potential sensitive fluorescent dyes may be used.
細胞膜は、膜電位感受性蛍光色素を適当な溶媒に溶解して調製した染色液を、例えば、３７℃の温度で細胞とともにインキュベートすることにより染色できる。このような染色により、膜電位感受性蛍光色素を、膜電位に応じて細胞膜外側表面及び細胞内に分布させることができる。染色液は、例えば、膜電位感受性蛍光色素をジメチルスルホキシド（ＤＭＳＯ）等の有機溶媒に溶解させ、さらにこれをHepes Buffer等の緩衝液で希釈することによって調製することができる。 The cell membrane can be stained by incubating a staining solution prepared by dissolving a membrane potential-sensitive fluorescent dye in a suitable solvent with the cells at a temperature of 37 ° C., for example. By such staining, the membrane potential sensitive fluorescent dye can be distributed on the outer surface of the cell membrane and in the cells according to the membrane potential. The staining solution can be prepared, for example, by dissolving a membrane potential sensitive fluorescent dye in an organic solvent such as dimethyl sulfoxide (DMSO) and further diluting it with a buffer solution such as Hepes Buffer.
細胞内又は細胞外媒体への蛍光消光色素の導入と、膜電位感受性蛍光色素による細胞の細胞膜の染色とは、同時に行ってもよく、いずれか一方を先に行ってもよいが、採用した手法に従って順番を決定することが好ましい。例えば、蛍光消光色素を浸透圧を変化させて水分とともにピノサイトーシスにより取り込ませる場合や、蛍光消光色素を電気穿孔により導入するような場合は、蛍光消光色素の導入を先に行うことが好ましい。色素を修飾して細胞内への移行性を高めた後、細胞とともにインキュベートする方法の場合は、細胞内への蛍光消光色素の導入と、膜電位感受性蛍光色素による細胞の細胞膜の染色とを、同時に行うことができる。細胞膜の染色後は、細胞を緩衝液で洗浄するのが好ましい。 The introduction of the fluorescence quenching dye into the intracellular or extracellular medium and the staining of the cell membrane of the cell with the membrane potential sensitive fluorescent dye may be performed simultaneously, or one of them may be performed first. It is preferable to determine the order according to For example, when the fluorescence quenching dye is changed by osmotic pressure to be taken in together with moisture by pinocytosis, or when the fluorescence quenching dye is introduced by electroporation, the fluorescence quenching dye is preferably introduced first. In the case of a method of incubating with cells after modifying the dye to improve the transferability into the cell, introduction of a fluorescence quenching dye into the cell and staining of the cell membrane of the cell with a membrane potential sensitive fluorescent dye, Can be done simultaneously. After staining of the cell membrane, the cells are preferably washed with a buffer solution.
測定ステップでは、導入ステップで得られた色素導入細胞に励起光を照射して、膜電位感受性蛍光色素から生じる蛍光の強度を経時的に測定する。蛍光強度は、例えば、励起光源（キセノンランプ、アルゴンレーザー等）と、励起光波長及び蛍光波長を選択する手段（フィルター）と、蛍光顕微鏡と、光検出器（ＣＣＤカメラ、光電子増倍管等）と、顕微鏡画像処理手段とを備える装置を用いて、一定のサンプリングインターバルで蛍光画像を取得することにより経時的に測定することができる。ウェルプレートのウェル内にある細胞の蛍光強度を測定する場合は、蛍光プレートリーダーを用いてもよい。蛍光消光色素が細胞外媒体に導入された場合には、蛍光強度の測定は、細胞を収容する容器の下方に光検出器を置いて行うことが好ましい。 In the measuring step, the dye-introduced cells obtained in the introducing step are irradiated with excitation light, and the intensity of the fluorescence generated from the membrane potential sensitive fluorescent dye is measured over time. The fluorescence intensity includes, for example, an excitation light source (xenon lamp, argon laser, etc.), means for selecting the excitation light wavelength and fluorescence wavelength (filter), a fluorescence microscope, and a photodetector (CCD camera, photomultiplier tube, etc.) And using a device including a microscope image processing means, it is possible to measure over time by acquiring a fluorescence image at a constant sampling interval. When measuring the fluorescence intensity of the cells in the well of the well plate, a fluorescence plate reader may be used. When the fluorescence quenching dye is introduced into the extracellular medium, it is preferable to measure the fluorescence intensity by placing a photodetector below the container containing the cells.
膜電位変化の検出は、蛍光強度の経時的測定の結果（蛍光強度の時間変化を示すグラフ）から行うこともできるが、測定開始時の蛍光強度を基準とした蛍光強度の最大変化量(以下場合により「蛍光強度の最大変化量」又は「最大変化量」という。)、並びに、測定時間−蛍光強度曲線の測定開始時から測定終了時までの積分値（以下場合により「積分値」という。）を求めてより定量的に行うのが好ましい。ここで、蛍光強度の最大変化量とは、絶対値が最大となる蛍光強度の変化量(負の場合もある)である。蛍光強度が図１のように変化した(測定時間はｔとする)とすると、図１において矢印で示した幅は最大変化量の大きさを、斜線部の面積は積分値の大きさを示す。蛍光強度が図１のように減少している場合は、最大変化量も積分値も負の値となる。 The detection of changes in membrane potential can be performed from the results of time-dependent measurement of fluorescence intensity (a graph showing the temporal change in fluorescence intensity), but the maximum amount of change in fluorescence intensity based on the fluorescence intensity at the start of measurement (hereinafter referred to as the fluorescence intensity change) In some cases, it is referred to as “maximum change amount of fluorescence intensity” or “maximum change amount”), and an integrated value from the start of measurement to the end of measurement of the measurement time-fluorescence intensity curve (hereinafter referred to as “integrated value” in some cases). ) Is preferably performed quantitatively. Here, the maximum amount of change in fluorescence intensity is the amount of change in fluorescence intensity that has the maximum absolute value (which may be negative). Assuming that the fluorescence intensity changes as shown in FIG. 1 (measurement time is t), the width indicated by the arrow in FIG. 1 indicates the maximum change amount, and the shaded area indicates the integral value. . When the fluorescence intensity decreases as shown in FIG. 1, both the maximum change amount and the integral value are negative values.
本発明の膜電位変化検出方法では、蛍光消光色素それ自体が蛍光性を有することにより生じるアーティファクトを検出するために、膜電位感受性蛍光色素による細胞膜の染色を行っていない細胞（蛍光消光色素は導入されている）についても蛍光強度を経時的に測定しておくことが好ましい。 In the method for detecting changes in membrane potential of the present invention, in order to detect artifacts caused by the fluorescence quenching dye itself having fluorescence, cells that have not been stained with a membrane potential sensitive fluorescence dye (fluorescence quenching dye is introduced). ) Is preferably measured over time.
蛍光強度の変化がアーティファクトでないと判断されれば、膜電位変化があったと判定することができる。例えば、蛍光消光色素が細胞内に導入された場合において、最大変化量及び積分値が負であれば、細胞膜上（外側表面）の膜電位感受性蛍光色素が減少したと判断でき、これにより膜電位の上昇を検出できる。また、蛍光消光色素が細胞外媒体に導入された場合において、最大変化量及び積分値が負であれば、細胞膜上（外側表面）の膜電位感受性蛍光色素が増加したと判断でき、これにより膜電位の下降を検出できる。 If it is determined that the change in fluorescence intensity is not an artifact, it can be determined that there has been a change in membrane potential. For example, when a fluorescence quenching dye is introduced into a cell, if the maximum amount of change and the integral value are negative, it can be determined that the membrane potential sensitive fluorescent dye on the cell membrane (outer surface) has decreased. Can be detected. In addition, when the fluorescence quenching dye is introduced into the extracellular medium, if the maximum change amount and the integral value are negative, it can be determined that the membrane potential sensitive fluorescent dye on the cell membrane (outer surface) has increased. A decrease in potential can be detected.
膜電位変化の検出においては、変化の程度だけでなく、膜電位が上昇後直ちに元に戻ったか、上昇したままであったかといった変化の態様も判定することができる。例えば、蛍光強度の最大変化量により膜電位変化の程度を判定することができるが、最大変化量が同じで膜電位変化が同程度であっても、同一時間内の積分値が異なれば、膜電位変化の態様が異なっていたことが分かる。 In the detection of the change in membrane potential, not only the degree of change but also the mode of change such as whether the membrane potential returned to its original state immediately after it rose or remained elevated can be determined. For example, the degree of change in membrane potential can be determined based on the maximum amount of change in fluorescence intensity. However, even if the maximum change is the same and the change in membrane potential is the same, if the integral value within the same time is different, the membrane It can be seen that the mode of potential change was different.
本発明の膜電位変化検出方法では、導入ステップ及び測定ステップの前に、細胞を培養するステップを設けることが好ましい。細胞を培養して増殖させれば、それだけ測定される蛍光強度が大きくなり、膜電位変化の検出感度も高くなる。 In the membrane potential change detection method of the present invention, it is preferable to provide a step of culturing cells before the introduction step and the measurement step. If cells are cultured and proliferated, the measured fluorescence intensity increases and the detection sensitivity of changes in membrane potential increases.
本発明の膜電位変化検出方法では、各ステップにおける操作を、細胞をウェルプレートのウェル内に収容させたまま行うのが好ましい。ウェルプレートの使用により、大量の検体を一度に処理することが可能となり、より高速・高効率の膜電位変化検出が可能となる。用いるウェルプレートは、２４ウェル、９６ウェル、３８４ウェル、１５３６ウェル等の各種ウェルプレートから目的に応じて選択することができる。 In the membrane potential change detection method of the present invention, the operation in each step is preferably performed while the cells are accommodated in the wells of the well plate. By using a well plate, it is possible to process a large amount of specimens at a time, and it is possible to detect a change in membrane potential more quickly and efficiently. The well plate to be used can be selected according to the purpose from various well plates such as 24 well, 96 well, 384 well, and 1536 well.
（薬物スクリーニング方法）
本発明の薬物スクリーニング方法は、前述の発現ステップ、導入ステップ、接触ステップ及び検出ステップを備えるものである。この方法における発現ステップでは、目的のイオンチャネル及び／又はイオン輸送体を細胞に発現させる。イオンチャネル又はイオン輸送体の細胞への発現は、目的のイオンチャネル又はイオン輸送体の遺伝子を発現ベクターに組み込み、これを細胞に導入することにより行うことができる。
(Drug screening method)
The drug screening method of the present invention comprises the aforementioned expression step, introduction step, contact step and detection step. In the expression step in this method, the target ion channel and / or ion transporter is expressed in the cell. Expression of the ion channel or ion transporter in the cell can be performed by incorporating the gene of the target ion channel or ion transporter into an expression vector and introducing the gene into the cell.
導入ステップでは、細胞内又は細胞外媒体への蛍光消光色素の導入、及び膜電位感受性蛍光色素による細胞膜の染色を、前述した膜電位変化検出方法の導入ステップと同様に行うことができる。 In the introduction step, introduction of the fluorescence quenching dye into the cell or extracellular medium and staining of the cell membrane with the membrane potential sensitive fluorescent dye can be performed in the same manner as in the introduction step of the membrane potential change detection method described above.
接触ステップでは、導入ステップで得られた色素導入細胞に候補薬物を接触させる。この接触は、候補薬物を適当な溶媒に溶解し、得られる溶液を色素導入細胞が収容された容器内部に添加することにより接触させることができる。候補薬物と色素導入細胞との接触は、次に説明する検出ステップにおける蛍光強度の測定中に行うのが好ましい。 In the contacting step, the candidate drug is brought into contact with the dye-introduced cell obtained in the introducing step. This contact can be made by dissolving the candidate drug in a suitable solvent and adding the resulting solution to the inside of the container containing the dye-introduced cells. The contact between the candidate drug and the dye-introduced cell is preferably performed during the measurement of the fluorescence intensity in the detection step described below.
検出ステップでは、導入ステップで得られた色素導入細胞に励起光を照射して、膜電位感受性蛍光色素から生じる蛍光の強度を経時的に測定することにより、候補薬物との接触による色素導入細胞の膜電位変化を検出する。蛍光強度の経時的測定は、前述した膜電位変化検出方法の測定ステップと同様に行うことができる。 In the detection step, the dye-introduced cells obtained in the introduction step are irradiated with excitation light, and the intensity of the fluorescence generated from the membrane potential-sensitive fluorescent dye is measured over time. A change in membrane potential is detected. The measurement of the fluorescence intensity over time can be performed in the same manner as the measurement step of the membrane potential change detection method described above.
膜電位変化の検出は、前述の膜電位変化検出方法と同様に、蛍光強度の経時的測定の結果（蛍光強度の時間変化を示すグラフ）から行うこともできるが、測定開始時の蛍光強度を基準とした蛍光強度の最大変化量、並びに、測定時間−蛍光強度曲線の測定開始時から測定終了時までの積分値を求めてより定量的に行うのが好ましい。 Membrane potential change can be detected from the result of time-dependent measurement of fluorescence intensity (a graph showing the temporal change in fluorescence intensity) as in the above-described method for detecting membrane potential change. It is preferable to carry out more quantitatively by obtaining the maximum change amount of the fluorescence intensity as a reference and the integrated value from the measurement start to the measurement end of the measurement time-fluorescence intensity curve.
本発明の薬物スクリーニング方法では、アーティファクトを検出するために、膜電位感受性蛍光色素による細胞膜の染色を行っていない細胞、候補薬物と接触させていない細胞、候補薬物とのみ接触させた細胞（候補薬物それ自体が蛍光性を有する場合）、又はイオンチャネル若しくはイオン輸送体を発現させていない細胞についても、蛍光強度を経時的に測定しておくことが好ましい。 In the drug screening method of the present invention, in order to detect artifacts, cells not stained with a membrane potential sensitive fluorescent dye, cells not contacted with a candidate drug, cells contacted only with a candidate drug (candidate drug) It is preferable that the fluorescence intensity is measured over time for cells that do not express an ion channel or an ion transporter).
蛍光強度の変化がアーティファクトでないと判断されれば、膜電位変化があったと判定でき、これにより、候補薬物が目的のイオンチャネル又はイオン輸送体に作用したと判定できる。 If it is determined that the change in fluorescence intensity is not an artifact, it can be determined that there has been a change in membrane potential, thereby determining that the candidate drug has acted on the target ion channel or ion transporter.
本発明の薬物スクリーニング方法では、発現ステップ後、導入ステップ及び測定ステップの前に、細胞を培養するステップを設けることが好ましい。細胞を培養して増殖させれば、それだけ測定される蛍光強度が大きくなり、膜電位変化の検出感度も高くなる。 In the drug screening method of the present invention, it is preferable to provide a step of culturing cells after the expression step and before the introduction step and the measurement step. If cells are cultured and proliferated, the measured fluorescence intensity increases and the detection sensitivity of changes in membrane potential increases.
本発明の薬物スクリーニング方法では、各ステップにおける操作を、細胞をウェルプレートのウェル内に収容させたまま行うのが好ましい。ウェルプレートの使用により、大量の検体を一度に処理することが可能となり、より高速・高効率の薬物スクリーニングが可能となる。用いるウェルプレートは、目的に応じて各種ウェルプレートから選択することができる。 In the drug screening method of the present invention, the operation in each step is preferably performed while the cells are accommodated in the wells of the well plate. The use of a well plate makes it possible to process a large amount of specimens at a time, enabling faster and more efficient drug screening. The well plate to be used can be selected from various well plates according to the purpose.
（膜電位変化検出用ウェルプレート）
本発明の膜電位変化検出用ウェルプレートは、ウェルプレートのウェルに収容された細胞の細胞内又は細胞外媒体に蛍光消光色素を導入し、かつその細胞の細胞膜を膜電位感受性蛍光色素で染色することにより作製することができる。用いるウェルプレートは、目的に応じて各種ウェルプレートから選択することができる。細胞内又は細胞外媒体への蛍光消光色素の導入、及び膜電位感受性蛍光色素による細胞膜の染色は、前述した膜電位変化検出方法におけるそれらの操作と同様に行うことができる。細胞内又は細胞外媒体への蛍光消光色素の導入、及び膜電位感受性蛍光色素による細胞膜の染色を行う前に、ウェルプレートのウェル内で細胞を培養しておくのが好ましい。
(Well plate for detecting membrane potential change)
The well plate for detecting a membrane potential change according to the present invention introduces a fluorescence quenching dye into the intracellular or extracellular medium of a cell accommodated in a well of the well plate, and stains the cell membrane of the cell with a membrane potential sensitive fluorescent dye. Can be produced. The well plate to be used can be selected from various well plates according to the purpose. Introduction of a fluorescence quenching dye into an intracellular or extracellular medium and staining of a cell membrane with a membrane potential sensitive fluorescent dye can be performed in the same manner as those operations in the above-described membrane potential change detection method. Before introducing the fluorescence quenching dye into the cell or the extracellular medium and staining the cell membrane with the membrane potential sensitive fluorescent dye, the cells are preferably cultured in the well of the well plate.
（薬物スクリーニング用ウェルプレート）
本発明の薬物スクリーニング用ウェルプレートは、ウェルプレートのウェルに収容された細胞に目的のイオンチャネル及び／又はイオン輸送体を発現させた後、この細胞内又は細胞外媒体に蛍光消光色素を導入し、かつその細胞の細胞膜を膜電位感受性蛍光色素で染色することにより作製することができる。用いるウェルプレートは、目的に応じて各種ウェルプレートから選択することができる。イオンチャネル及び／又はイオン輸送体の細胞への発現、細胞内又は細胞外媒体への蛍光消光色素の導入、及び膜電位感受性蛍光色素による細胞膜の染色は、前述した膜電位変化検出方法又は薬物スクリーニング方法におけるそれらの操作と同様に行うことができる。細胞内又は細胞外媒体への蛍光消光色素の導入、及び膜電位感受性蛍光色素による細胞膜の染色を行う前に、ウェルプレートのウェル内で細胞を培養しておくのが好ましい。
(Well plate for drug screening)
In the well plate for drug screening of the present invention, a target ion channel and / or ion transporter is expressed in cells contained in the well of the well plate, and then a fluorescence quenching dye is introduced into the intracellular or extracellular medium. In addition, the cell membrane of the cell can be prepared by staining with a membrane potential sensitive fluorescent dye. The well plate to be used can be selected from various well plates according to the purpose. Expression of an ion channel and / or ion transporter into a cell, introduction of a fluorescence quenching dye into an intracellular or extracellular medium, and staining of a cell membrane with a membrane potential sensitive fluorescent dye may be performed by the above-described membrane potential change detection method or drug screening. It can be performed in the same manner as those operations in the method. Before introducing the fluorescence quenching dye into the cell or the extracellular medium and staining the cell membrane with the membrane potential sensitive fluorescent dye, the cells are preferably cultured in the well of the well plate.
以下、本発明の実施例を説明するが、本発明はこれらの実施例に限定されない。 Examples of the present invention will be described below, but the present invention is not limited to these examples.
（実施例１）
細胞の培養：
ヒト血管平滑筋細胞を９６ウエルマイクロプレート（Nulgenunc, 165305）に１ｘ１０Ｅ５ cells／wellで蒔き、１０％ウシ胎児血清（ＦＢＳ）（GIBCO）を添加したＤＭＥＭ／Ｆ１２（GIBCO, 11320-033）で２４時間培養した。
Example 1
Cell culture:
Human vascular smooth muscle cells were seeded in a 96-well microplate (Nulgenunc, 165305) at 1 × 10E5 cells / well for 24 hours in DMEM / F12 (GIBCO, 11320-033) supplemented with 10% fetal bovine serum (FBS) (GIBCO). Cultured.
蛍光消光色素及び膜電位感受性蛍光色素の負荷：
蛍光消光色素としては、波長５００ｎｍ〜６００ｎｍに幅広い吸収域を持つアマランス（Sigma, A1016）を用いた。アマランスは、培養液（ＤＭＥＭ／Ｆ１２＋１０％ＦＢＳ）に溶解して１μｇ／ｍｌ溶液とした。色素導入キットのプロトコールに従って、Pinocytic Cell-Loading Reagent（Molecular Probes, I-14402）でヒト血管平滑筋細胞内の浸透圧を変化させ、アマランスを細胞に取り込ませた。
Fluorescent quenching dye and membrane potential sensitive fluorescent dye loading:
As the fluorescence quenching dye, Amaranth (Sigma, A1016) having a wide absorption range at a wavelength of 500 nm to 600 nm was used. Amaranth was dissolved in a culture solution (DMEM / F12 + 10% FBS) to give a 1 μg / ml solution. According to the protocol of the dye introduction kit, the osmotic pressure in human vascular smooth muscle cells was changed with Pinocytic Cell-Loading Reagent (Molecular Probes, I-14402), and amaranth was incorporated into the cells.
膜電位感受性蛍光色素としては、DiSBAC2(3)（Molecular Probes, B-413）を用いた。DiSBAC2(3)をＤＭＳＯに溶解し、Hanks-Hepes Buffer（２０ｍＭ Hepes, １１５ｍＭＮａＣｌ, ５．４ｍＭ ＫＣｌ, ０．８ｍＭ ＭｇＣｌ２, １．８ｍＭ ＣａＣｌ２, １３．８ｍＭ D-Glucose）を加えて５μＭの染色液を作製した。これを、消光色素を取り込ませたヒト血管平滑筋細胞に加え、３７℃で３０分間インキュベートした。その後、ヒト血管平滑筋細胞をHanks-Hepes Bufferで２回洗浄し、最終的に１００μl／wellのHanks-Hepes Bufferをウェルに加えた。 DiSBAC 2 (3) (Molecular Probes, B-413) was used as a membrane potential sensitive fluorescent dye. DiSBAC 2 (3) was dissolved in DMSO, Hanks-Hepes Buffer (20 mM Hepes, 115 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl 2 , 1.8 mM CaCl 2 , 13.8 mM D-Glucose) was added to add 5 μM. A staining solution was prepared. This was added to human vascular smooth muscle cells incorporating a quenching dye, and incubated at 37 ° C. for 30 minutes. Thereafter, human vascular smooth muscle cells were washed twice with Hanks-Hepes Buffer, and finally 100 μl / well Hanks-Hepes Buffer was added to the wells.
蛍光強度の測定：
蛍光強度測定装置としては、倒立型蛍光顕微鏡ＩＸ−７１（オリンパス）に、７５Ｗキセノンランプと励起光波長４８０ｎｍ・蛍光波長５４０ｎｍ以上のフィルターをセットし、そこに冷却ＣＣＤカメラ（ＯＲＣＡ−ＥＲ、浜松ホトニクス）を搭載した顕微鏡画像処理装置AQUACOSMOS（浜松ホトニクス）を取り付けたものを用いた。
Measurement of fluorescence intensity:
As a fluorescence intensity measurement device, an inverted fluorescence microscope IX-71 (Olympus) is set with a 75 W xenon lamp and a filter having an excitation light wavelength of 480 nm and a fluorescence wavelength of 540 nm or more, and a cooled CCD camera (ORCA-ER, Hamamatsu Photonics). ) Equipped with a microscope image processing device AQUACOSMOS (Hamamatsu Photonics).
蛍光消光色素及び膜電位感受性蛍光色素を負荷したヒト血管平滑筋細胞の入った９６ウェルマイクロプレートを顕微鏡ステージにセットし、１秒間のサンプリングインターバルで２分間蛍光画像を取り込んで、経時的に蛍光強度を測定した。測定開始から１０秒後、６００ｍＭＫＣｌ溶液（終濃度２００ｍＭ）（５０μｌ）、１５０ｍＭ ＫＣｌ溶液（終濃度５０ｍＭ）（５０μｌ）、７５ｍＭＫＣｌ溶液（終濃度２５ｍＭ）（５０μｌ）、３０ｍＭ ＫＣｌ溶液（終濃度１０ｍＭ）（５０μｌ）、０ｍＭＫＣｌ溶液（終濃度０ｍＭ）（５０μｌ）を分注した。ここで、ＫＣｌ溶液は、細胞膜を脱分極させるために加えたものである。表１は、測定開始時の蛍光強度を１．０としたときの、加えたＫＣｌ溶液の濃度ごとの最大変化量及び積分値を示す。
A 96-well microplate containing human vascular smooth muscle cells loaded with fluorescent quenching dye and membrane potential sensitive fluorescent dye is set on a microscope stage, and a fluorescent image is captured for 2 minutes at a sampling interval of 1 second. Was measured. 10 seconds after the start of measurement, 600 mM KCl solution (final concentration 200 mM) (50 μl), 150 mM KCl solution (final concentration 50 mM) (50 μl), 75 mM KCl solution (final concentration 25 mM) (50 μl), 30 mM KCl solution (final concentration 10 mM) ( 50 μl) and 0 mM KCl solution (
（実施例２）
細胞の培養：
細胞の培養は、実施例１と同様に行った。
(Example 2)
Cell culture:
Cell culture was carried out in the same manner as in Example 1.
蛍光消光色素及び膜電位感受性蛍光色素の負荷：
蛍光消光色素としては、ポリフェノール（フラボノイド系）の一種である青色アントシアニンをエステル化したものを用いた。
Fluorescent quenching dye and membrane potential sensitive fluorescent dye loading:
As the fluorescence quenching dye, an esterified blue anthocyanin which is a kind of polyphenol (flavonoid type) was used.
膜電位感受性蛍光色素としては、di-8-ANEPPS（Molecular Probes, D-3167）を用いた。di-8-ANEPPSをＤＭＳＯに溶解し、Hanks-Hepes Buffer（２０ｍＭ Hepes, １１５ｍＭＮａＣｌ, ５．４ｍＭ ＫＣｌ, ０．８ｍＭ ＭｇＣｌ２, １．８ｍＭ ＣａＣｌ２, １３．８ＭＭ D-Glucose）を加えて５μＭの染色液を作製した。 Di-8-ANEPPS (Molecular Probes, D-3167) was used as a membrane potential sensitive fluorescent dye. Dissolve di-8-ANEPPS in DMSO and add Hanks-Hepes Buffer (20 mM Hepes, 115 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl 2 , 1.8 mM CaCl 2 , 13.8MM D-Glucose) to add 5 μM. A staining solution was prepared.
蛍光消光色素の終濃度が１００μｇ／ｍｌになるように蛍光消光色素のＤＭＳＯ溶液を染色液と混合し、これをヒト血管平滑筋細胞に加えて３７℃で３０分間インキュベートした。その後、ヒト血管平滑筋細胞をHanks-Hepes Bufferで２回洗浄し、最終的に１００μl／wellのHanks-Hepes Bufferをウェルに加えた。 A DMSO solution of a fluorescence quenching dye was mixed with a staining solution so that the final concentration of the fluorescence quenching dye was 100 μg / ml, and this was added to human vascular smooth muscle cells and incubated at 37 ° C. for 30 minutes. Thereafter, human vascular smooth muscle cells were washed twice with Hanks-Hepes Buffer, and finally 100 μl / well Hanks-Hepes Buffer was added to the wells.
蛍光強度の測定：
蛍光強度の測定は、実施例１と同様に行った。表２は、測定開始時の蛍光強度を１．０としたときの、加えたＫＣｌ溶液の濃度ごとの最大変化量及び積分値を示す。
Measurement of fluorescence intensity:
The fluorescence intensity was measured in the same manner as in Example 1. Table 2 shows the maximum change amount and integrated value for each concentration of the added KCl solution when the fluorescence intensity at the start of measurement is 1.0.
実施例１及び２の結果（表１及び２）から、本発明の膜電位変化検出方法により膜電位変化を高感度かつ簡便に検出することができることが明らかとなった。 From the results of Examples 1 and 2 (Tables 1 and 2), it became clear that the membrane potential change can be detected with high sensitivity and simplicity by the membrane potential change detection method of the present invention.
本発明は、膜電位に関わる生化学・生理学的研究や種々の薬物（イオンチャネル作用薬、抗生物質等）の薬効の評価に利用することができる。特に、イオンチャネル作用薬やイオン輸送体作用薬のハイスループットスクリーニングにおいて有効である。 INDUSTRIAL APPLICABILITY The present invention can be used for biochemical / physiological studies relating to membrane potential and evaluation of the efficacy of various drugs (ion channel agonists, antibiotics, etc.). In particular, it is effective in high-throughput screening of ion channel agonists and ion transporter agonists.
Claims (6)
前記色素導入細胞に励起光を照射して、前記膜電位感受性蛍光色素から生じる蛍光の強度を経時的に測定するステップと、を備える膜電位変化検出方法。 Introducing a fluorescence quenching dye into an intracellular or extracellular medium and staining the cell membrane of the cell with a membrane potential-sensitive fluorescent dye, either simultaneously or in advance to obtain a dye-introduced cell;
Irradiating excitation light to the dye-introduced cell and measuring the intensity of fluorescence generated from the membrane potential-sensitive fluorescent dye over time.
前記色素導入細胞に励起光を照射して、前記膜電位感受性蛍光色素から生じる蛍光の強度を経時的に測定し、測定開始時の蛍光強度を基準とした蛍光強度の最大変化量、並びに、測定時間−蛍光強度曲線の測定開始時から測定終了時までの積分値、を求めるステップと、を備える膜電位変化検出方法。 Introducing a fluorescence quenching dye into an intracellular or extracellular medium and staining the cell membrane of the cell with a membrane potential-sensitive fluorescent dye, either simultaneously or in advance to obtain a dye-introduced cell;
Irradiate excitation light to the dye-introduced cells, measure the intensity of fluorescence generated from the membrane potential-sensitive fluorescent dye over time, and measure the maximum amount of change in fluorescence intensity based on the fluorescence intensity at the start of measurement, as well as measurement A method for detecting a change in membrane potential, comprising: calculating an integral value from the start of measurement to the end of measurement of the time-fluorescence intensity curve.
前記イオンチャネル及び／又はイオン輸送体を前記細胞に発現させるステップと、
前記細胞内又は細胞外媒体への蛍光消光色素の導入と、膜電位感受性蛍光色素による前記細胞の細胞膜の染色とを、同時に又はいずれか一方を先に実施して色素導入細胞を得るステップと、
候補薬物を前記色素導入細胞に接触させるステップと、
前記色素導入細胞に励起光を照射して、前記膜電位感受性蛍光色素から生じる蛍光の強度を経時的に測定することにより、前記候補薬物との接触による前記色素導入細胞の膜電位変化を検出するステップと、を備えるスクリーニング方法。 A screening method for drugs that act on ion channels and / or ion transporters of cells,
Expressing the ion channel and / or ion transporter in the cell;
Introducing a fluorescence quenching dye into the intracellular or extracellular medium, and staining the cell membrane of the cell with a membrane potential sensitive fluorescent dye, either simultaneously or in advance to obtain a dye-introduced cell;
Contacting a candidate drug with the dye-introduced cell;
By irradiating the dye-introduced cell with excitation light and measuring the intensity of fluorescence generated from the membrane potential-sensitive fluorescent dye over time, a change in the membrane potential of the dye-introduced cell due to contact with the candidate drug is detected A screening method comprising the steps of:
前記イオンチャネル及び／又はイオン輸送体を前記細胞に発現させるステップと、
前記細胞内又は細胞外媒体への蛍光消光色素の導入と、膜電位感受性蛍光色素による前記細胞の細胞膜の染色とを、同時に又はいずれか一方を先に実施して色素導入細胞を得るステップと、
候補薬物を前記色素導入細胞に接触させるステップと、
前記色素導入細胞に励起光を照射して、前記膜電位感受性蛍光色素から生じる蛍光の強度を測定し、測定開始時の蛍光強度を基準とした蛍光強度の最大変化量、並びに、測定時間−蛍光強度曲線の測定開始時から測定終了時までの積分値、を求めることにより、前記候補薬物との接触による前記色素導入細胞の膜電位変化を検出するステップと、を備えるスクリーニング方法。 A screening method for drugs that act on ion channels and / or ion transporters of cells,
Expressing the ion channel and / or ion transporter in the cell;
Introducing a fluorescence quenching dye into the intracellular or extracellular medium, and staining the cell membrane of the cell with a membrane potential sensitive fluorescent dye, either simultaneously or in advance to obtain a dye-introduced cell;
Contacting a candidate drug with the dye-introduced cell;
The dye-introduced cells are irradiated with excitation light, the intensity of fluorescence generated from the membrane potential-sensitive fluorescent dye is measured, the maximum amount of change in fluorescence intensity based on the fluorescence intensity at the start of measurement, and the measurement time-fluorescence Detecting a change in membrane potential of the dye-introduced cell due to contact with the candidate drug by obtaining an integrated value from the start of the measurement of the intensity curve to the end of the measurement.
前記イオンチャネル及び／又はイオン輸送体が発現し、細胞内又は細胞外媒体への蛍光消光色素の導入と、膜電位感受性蛍光色素による細胞膜の染色とが行われた細胞を、ウェルプレートのウェル内に備えるウェルプレート。
A well plate for screening for drugs that act on ion channels and / or ion transporters of cells,
Cells that express the ion channel and / or ion transporter and have undergone introduction of a fluorescence quenching dye into a cell or an extracellular medium and staining of the cell membrane with a membrane potential sensitive fluorescent dye are transferred into the well of the well plate. Well plate to prepare for.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2004316512A JP4421445B2 (en) | 2004-10-29 | 2004-10-29 | Membrane potential change detection method, drug screening method, and well plate |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2004316512A JP4421445B2 (en) | 2004-10-29 | 2004-10-29 | Membrane potential change detection method, drug screening method, and well plate |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2006126073A true JP2006126073A (en) | 2006-05-18 |
| JP4421445B2 JP4421445B2 (en) | 2010-02-24 |
Family
ID=36720951
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2004316512A Active JP4421445B2 (en) | 2004-10-29 | 2004-10-29 | Membrane potential change detection method, drug screening method, and well plate |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP4421445B2 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2011007741A (en) * | 2009-06-29 | 2011-01-13 | Nippon Telegr & Teleph Corp <Ntt> | Odor sensor, and odor detecting method |
| WO2012002460A1 (en) | 2010-06-29 | 2012-01-05 | 公立大学法人名古屋市立大学 | Material for screening for compound capable of acting on ion channel, and use thereof |
| WO2018084221A1 (en) | 2016-11-01 | 2018-05-11 | 公立大学法人名古屋市立大学 | Material for screening compounds acting on ion channels and use of said material |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR102400237B1 (en) * | 2020-08-04 | 2022-05-23 | 재단법인대구경북과학기술원 | Screening device for electroceutical and method using thereof |
-
2004
- 2004-10-29 JP JP2004316512A patent/JP4421445B2/en active Active
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2011007741A (en) * | 2009-06-29 | 2011-01-13 | Nippon Telegr & Teleph Corp <Ntt> | Odor sensor, and odor detecting method |
| WO2012002460A1 (en) | 2010-06-29 | 2012-01-05 | 公立大学法人名古屋市立大学 | Material for screening for compound capable of acting on ion channel, and use thereof |
| US9133496B2 (en) | 2010-06-29 | 2015-09-15 | Public University Corporation Nagoya City University | Material for screening for compound acting on ion channel and use thereof |
| WO2018084221A1 (en) | 2016-11-01 | 2018-05-11 | 公立大学法人名古屋市立大学 | Material for screening compounds acting on ion channels and use of said material |
| US11828750B2 (en) | 2016-11-01 | 2023-11-28 | Public University Corporation Nagoya City University | Material for screening for compound acting on ION channel and use thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| JP4421445B2 (en) | 2010-02-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20220021823A1 (en) | Digitally enhanced microscopy for multiplexed histology | |
| Digman et al. | Mapping the number of molecules and brightness in the laser scanning microscope | |
| Zanetti-Domingues et al. | Hydrophobic fluorescent probes introduce artifacts into single molecule tracking experiments due to non-specific binding | |
| JP2023067901A (en) | High-throughput and highly multiplexed imaging with programmable nucleic acid probes | |
| EP2833140B1 (en) | Method for detecting biological material | |
| US20200200740A1 (en) | Method for detecting extracellular vesicles in a sample | |
| Meyer-Almes | Fluorescence lifetime based bioassays | |
| JP2006194711A (en) | Performance evaluation method and performance evaluation device of fluorescence-emission pigment | |
| CN107389636A (en) | A kind of preparation and application of the water soluble fluorescence sensor that endogenous glutathione can be detected in cancer cell | |
| Saha et al. | Dynamics of the HIV gag lattice detected by localization correlation analysis and time-lapse IPALM | |
| US20130137127A1 (en) | Dating bloodstains and biological fluids with fluorescence lifetime techniques | |
| JP4421445B2 (en) | Membrane potential change detection method, drug screening method, and well plate | |
| Ruedas-Rama et al. | FLIM Strategies for Intracellular Sensing: Fluorescence Lifetime Imaging as a Tool to Quantify Analytes of Interest | |
| Verma et al. | Detection and identification of amino acids and proteins using their intrinsic fluorescence in the visible light spectrum | |
| CN105466902A (en) | Method for measuring fluorescence resonance energy transfer sensitized quenching conversion factors | |
| Chen et al. | Probing nucleocytoplasmic transport by two‐photon activation of PA‐GFP | |
| Crystall et al. | Time resolved evanescent wave induced fluorescence measurements of surface adsorbed bovine serum albumin | |
| Luo et al. | Discrimination of wet or dried arterial and venous blood for forensic applications via eosin fluorescence lifetime | |
| JP2008249433A (en) | Method for measuring bonding affinity of probe to test material, and its utilization | |
| JP6017724B1 (en) | Method for evaluating the suitability of duodenal juice samples as pancreatic juice-derived components detection samples | |
| WO2006003397A2 (en) | Membrane fusion assay | |
| KR102315849B1 (en) | Detection method using step-wise on/off detection of biological targets, detection system and its manufacturing method | |
| Ciepla et al. | trLRET microscopy: Ultrasensitive imaging of lanthanide luminophores | |
| Gest et al. | A red-emitting carborhodamine for monitoring and measuring membrane potential | |
| Aurousseau et al. | A Step-by-Step Guide to Single-Subunit Counting of Membrane-Bound Proteins in Mammalian Cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20070515 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20090804 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20091005 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20091201 |
|
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20091202 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20121211 Year of fee payment: 3 |
|
| R150 | Certificate of patent or registration of utility model |
Free format text: JAPANESE INTERMEDIATE CODE: R150 Ref document number: 4421445 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20131211 Year of fee payment: 4 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |