JP2883149B2 - Viable cell counting method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Object of the invention]

[Industrial Application Field] The present invention relates to a method for simply and reliably measuring the number of living cells or the degree of vitality of living cells or living tissues or organs containing living cells. [Prior art] Conventionally, as a method for measuring living cells, there is a method in which living cells and dead cells are distinguished by cell staining, and the number of living cells is measured under a microscope. Because of the differences, discrimination requires empirical training and is not suitable for examining many samples. As another method, there is a method in which a living cell is stained with neutral red, and then the degree of staining is measured using a colorimeter. However, since it takes several hours for the living cell to take in the dye, the method lacks rapidity. As a further alternative, trace amounts of ATP contained in living cells
Although there are methods for measuring the amount of NAD (P) H and NAD (P) H, it is hardly a practical method in view of the number of steps required to extract the compound, preparation of an enzyme for measurement, and strict cleanliness requirements for instruments. In view of the above circumstances, the present inventors have previously proposed a method of measuring the amount of hydrogen peroxide generated when quinone is added to living cells using chemiluminescence (Japanese Patent Application Laid-Open No. 1-169499). Since the period is only about 5 seconds, it was necessary to form a strict measurement system for accurate measurement. [Problems to be Solved by the Invention] Accordingly, the present invention non-destructively performs quantitative measurement on living cells by devising a reaction system for continuously emitting light from living cells or living tissues simply by adding a drug. It is intended to provide a means for quick implementation.

Configuration of the Invention

 [Means for Solving the Problems] (1) Concept The present inventor has been a problem in the above-mentioned prior invention method.
Various studies have been made on methods to enhance the stability of luminescence.
As a result, oxidized quinone and transition metal compounds
Addition of luminol and luminol causes sustained chemiluminescence
Here, the intensity of this luminescence correlates with the number or amount of viable cells.
I found the fruit. (2) Outline Based on the above findings, the present invention provides a measurement system including living cells.
Oxidized quinone, transition metal compound and luminol
Live cell measurement characterized by measuring luminescence intensity
The method is summarized. The following items related to the configuration of the invention
It is described separately. (3) Luminescence reaction mechanism Luminol (5-amino-2,3-dihydro-1,4-lid
Razinedione; 3-aminophthalic hydrazide) (the following formula
I) is the most important chemiluminescent substance and its luminescence phenomenon
Are contacted by iron, copper, etc., but the mechanism is complicated.
You. The generally considered mechanism is as follows:
6-membered peroxide via diazaquinoline (Formula II)
The compound forms and emits light upon decomposition. And this
The oxygen molecules involved in the reaction of_TwoO_TwoI think
It is imaged. The present inventors have proposed that an oxidized quino such as menadione be added to a living cell system.
Of iron and EDTA (ethylenediaminetetraacetic acid) complex salts
Immediately persists when metal transfer compound and luminol are added
Was observed to produce luminescent light, but O
(Superoxide anion)
Eliminate superoxide dismutase or hydrogen peroxide
Has the property of promoting the oxidation of catalase or iron
Addition of ceruloplasmin inhibits luminescence
The emission is simply basic H_TwoO_TwoNot only by
O And iron are also presumed to play a concerted role in luminescence.
It is. And the formation of these oxygen molecules and reduced EDTA
Iron formation is catalyzed by quinone reduced by cells.
In addition, reduction of oxidized quinone requires intracellular NAD
(P) H: Involving quinone (namedione) oxidoreductase
It is supposed to be. (4) Quinone The quinone in the present invention includes benzoquinone and naphthoquino.
And quinones such as diphenoquinone and anthraquinone;
Including those derivatives, but easily oxidizable other than quinone group
Those having no portion are preferred. Representative quinone above
Can be used for the purpose of the invention, but they are physiological substances.
Particularly suitable is vitamin K_Three2 known as
-Methyl-1,4-naphthoquinone (menadione). (5) Transition metal compound As the transition metal compound in the present invention, for example, Fe,
Water-soluble compounds of transition metal elements such as Cu, Ni, Co, Mn, Ti and V
Products, but in general, for example, iron chloride, iron sulfate
It is desirable to use divalent water-soluble iron salts such as iron nitrate.
No. And preferably aminodicarboxylic acid (eg EDTA)
It is preferable to block with a quinolating agent such as
You. (Application) As is clear from the above principle, the measuring method of the present invention
As long as cell membrane-localized oxidoreductase is present,
Microorganisms, plants, which have the ability to oxidize luminol in their presence
Widely applicable to animal cells and tissues. For example, plant
Contains luminol, quinone, and transition metal compound
Luminescence is observed only by immersion in alkaline aqueous solution, no treatment
The root vitality can be measured as it is. Animals in tissue culture
It can also be applied to cell growth status and viable cell count measurement, especially for cancer
Effective for determining in vitro efficacy of anticancer agents on cells
Applicable to [Action] In the present invention, quinone reduced by living cells
Acts on transition metal compounds and oxygen molecules and is necessary for chemiluminescence.
Form molecular species. Between this luminescence intensity and the number of living cells
Since there is a correlation, measure the emission intensity
Allows simple and quick viable cell counts
You. [Examples] Hereinafter, embodiments of the present invention will be described with reference to examples.
Is for illustrative purposes only and is not intended to limit the inventive concept.
Absent. Example (FIG. 1) Yeast was added to 900 μl of 0.2 M Tris / HCl buffer (pH 9.5).
The viable cell density is 1.7X10⁷It was adjusted to pieces / ml. Incubate the prepared cell suspension at 25 ° C and add 50 μl
Aqueous solution (6 μg / ml) and 25 μl of 20 mM menadione
Increase of luminescence was observed when alcohol solution was added
Then, further add 10 μl of 10 mM ferrous chloride and 10 mM aqueous EDTA.
In addition, the emission intensity was amplified about 1000 times (see Fig. 1).
See). From this, luminol luminescence is comparable to menadione
It can be seen that both are significantly enhanced by the iron compound. Example 2 (FIG. 2) In the experiment of the previous example, the pH was changed by fixing conditions other than pH.
The relationship between chemical conversion and emission intensity was examined. As shown in FIG.
9.5 was found to be the optimal condition. In another experiment, chemiluminescence was observed in order to
As a final concentration, the amount of iron-EDTA complex salt should be 0.02 mM or more,
For dione, 0.1 mM or more, for luminol, 6 μg / ml or more
Each is necessary. Example 3 (FIG. 3) Only the density of yeast was changed under the conditions of Example 1,
The correlation with the emission intensity was examined. As a result, as shown in FIG.
Sea urg, 8.8x10^FourPieces / ml to 8.8x10⁶Between the number of bacteria / ml
It has been found that there is a proportional relationship with light intensity. What
8.8x10 by suppressing the emission noise^ThreeMeasured to pieces / ml
Was possible.

【The invention's effect】

As described above, the present invention can contribute to bio-related research and development and industrial development by providing a means for inexpensively and quickly performing quantitative measurement on living cells.

[Brief description of the drawings]

FIG. 1 is a graph showing the effect of luminol, menadione and an iron-EDTA complex on the luminescence intensity of living yeast cells.
The figure is a graph showing the effect of pH on the luminescence intensity of living yeast cells, and FIG. 3 is a graph showing the correlation between the number of living yeast cells and the luminescence intensity. The parameters of each figure are already described in each figure.

──────────────────────────────────────────────────続 き Continued on the front page (58) Fields surveyed (Int. Cl. ⁶ , DB name) C12Q 1/06 BIOSIS (DIALOG) WPI (DIALOG)

Claims (2)

(57) [Claims] 1. An oxidized quinone, iron,
A method for measuring live cells, comprising measuring the intensity of luminescence by adding an EDTA complex salt and luminol. 2. The method according to claim 1, wherein namedion is used as the quinone.