KR20200078239A - A Method for Measuring Intracellular pH - Google Patents

Abstract

The present invention relates to a method for measuring intracellular pH. More specifically, the present invention relates to a method for improving accuracy of intracellular pH measurement by controlling and treating a concentration of a dye capable of displaying fluorescence in cells used for measuring the intracellular pH based on the number of cells to be subject to pH measurement. The method for measuring the intracellular pH includes a step of treating the cells to be subject to pH measurement with the dye which exhibits the fluorescence in the cells.

Description

A method for measuring intracellular pH

The present invention relates to a method for measuring intracellular pH, and specifically, the concentration of a dye capable of exhibiting fluorescence in a cell used to measure intracellular pH is controlled by adjusting the number of cells to be measured based on the number of cells to be measured, and thus intracellular pH It relates to a method for improving the accuracy of the measurement.

Cells can be cultured by sowing in plastic containers, glass dishes or flasks. In order to maintain the acidity (pH) suitable for cell culture, the culture medium is exchanged. Due to the importance associated with life support, the pH of the intracellular environment is tightly regulated through various ion channels such as alkali cation-H+ exchangers, bicarbonates and acid load carriers, and weak acids and bases in the cell.

The pH in cells can be judged to be an important marker for identifying changes in cancer cells and the like. pH is one of the factors that play an important role in initiating and regulating numerous cellular activities such as multi-drug resistance in tumors, protein processing, endocytosis and cell death.

Separate pH values are maintained to maintain optimal operating conditions for specific metabolic functions in mammalian cells. Under normal physiological conditions, the resting cell pH of mammalian cells can be maintained between 6.8 and 7.3. On the other hand, the extracellular pH value is maintained in the range of 7.2 to 7.4 to exhibit weak alkalinity.

Impaired regulation of intracellular pH is often associated with changes in cell function, proliferation and drug resistance, and can be observed in cancer or tumors. In addition, when a disorder occurs in intracellular pH regulation, it has a great influence on tumor growth and cancer cell migration, so there is a possibility of cancer metastasis.

Anaerobic metabolism in which the extracellular pH level is reduced to less than 6.0 is induced by locally activated metabolism, and aerobic metabolism occurs that increases the intracellular concentration of carbon dioxide (CO ₂ ) to reduce the local pH level. It is also done.

Such intracellular pH changes affect the development, growth, and migration of various tumor cells or cancer cells, and a process of confirming whether these intracellular pH changes in cancer research may be essential.

In addition, as a number of new drug screening, drug delivery systems, etc. are performed using pH sensitive polymers or pH sensitive polymer nanoparticles, more precise measurement and analysis of intracellular pH data can affect the development of new drugs or carriers. And may act as a marker to determine whether or not the drug works effectively to the treatment of the disease.

Thus, it may be important to quantitatively measure intracellular pH and improve the accuracy of the pH measurement.

Meanwhile, various pH conditions in the cell may be measured through fluorescence. Fluorescence is the most commonly used technique for non-destructively tracking or analyzing biological molecules. By analyzing changes in various chemical and optical properties, such as simple luminescence properties, imaging using changes in luminescence properties, energy transfer, changes in luminescence properties due to changes in the surrounding environment, and changes in luminescence properties due to structural changes in response to chemical reactions, the life science field Useful information can be obtained.

However, since the fluorescence intensity is difficult to quantify directly, it can be indirectly confirmed through the measurement of the position of the dye, the excitation wavelength, etc., but it may be difficult to accurately measure it because it is influenced by experimental factors caused by cells according to the rate of cell absorption and release. there is a problem.

Under this technical background, the inventors of the present application can improve the accuracy of intracellular pH measurement by adjusting the concentration of a dye that exhibits fluorescence in the cell to be treated for intracellular pH measurement based on the number of cells to be measured for pH. It confirmed, and completed this invention.

In order to solve the above problems, the present invention provides a method for improving the accuracy of intracellular pH measurement.

In order to achieve the above object, the present invention is a method for improving the accuracy of intracellular pH measurement, comprising the step of treating a dye that exhibits fluorescence in cells in a cell to be measured for pH, and the concentration of the dye is measured at pH It provides a method characterized in that the number of target cells is counted and adjusted based on the number of cells.

According to the present invention, in the process of measuring the intracellular pH by treating the dye that exhibits fluorescence in the cell, when the same amount of dye is treated regardless of the number of cells, the problem that the pH cannot be accurately measured is solved, and the pH is measured. The intracellular pH can be accurately measured regardless of the number of cells.

1 to 3 show the results of measuring pH by treating BCECF-AM in proportion to the number of cells in the breast cancer cell line MDA-MB-231.
4 shows statistical values for results measured in FIGS. 1 to 3.
5 to 7 show the results of measuring the pH by treating BCECF-AM in proportion to the number of cells in the Korean colorectal cancer cell line SNU81.
8 shows statistical values for the results measured in FIGS. 5 to 7.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

In one aspect, the present invention is a method for improving the accuracy of intracellular pH measurement, comprising the step of treating a dye that exhibits fluorescence in the cell to the pH measurement cell, the concentration of the dye is the number of cells to be measured pH It relates to a method characterized in that by counting and adjusting based on the number of cells.

The dye that exhibits fluorescence in the cell may be, for example, a material that penetrates the cell membrane and is converted into a fluorescent material in the cell to exhibit fluorescence. The fluorescent material is a material that generates light by changing physical conditions and chemical treatment. According to the present invention, the intensity of light generated according to a change in pH within a cell may be changed.

The fluorescent dye in the cell is, for example, BCECF-AM(2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester), calcein-AM (Calcein) -acetoxymethyl ester), fura-2-AM(Fura-2-acetoxymethyl ester), indo-1-acetoxymethyl ester (Indo-1-AM), indo-5F-acetoxymethyl ester (India-5F-AM), queen-2 -AM(Quin-2-acetoxymethyl ester), 5-CFDA-AM(5-Carboxyfluorescein Diacetate-acetoxymethyl ester), BAPTA-AM(bis(2-aminophenoxy)ethane tetraacetic acid-acetoxymethyl ester), 5,5'-di Fluoro BAPTA-AM (5,5'-difluoro BAPTA-AM), 5,5'-dimethyl BAPTA-AM (5,5'-dimethyl BAPTA-AM), 5,5'-Dinitro BAPTA-AM (5 ,5'-dinitro BAPTA-AM), dihydrocalcein-acetoxymethyl ester (AM), EGTA-acetoxymethyl ester (EGTA-AM), Fluo-3-acetoxymethyl ester (Fluo-3-AM), Fluo-8 -AM(Fluo-8-acetoxymethyl ester), Rod-2-AM(Rhod-2-acetoxymethyl ester), Rod-4-AM(Rhod-2-acetoxymethyl ester), Rod-5F-AM(Rhod-5F-acetoxymethyl ester), rod-5N-AM (Rhod-5N-acetoxymethyl ester), X-rod-1-AM (X-Rhod-1-acetoxymethyl ester), and the like, but is not limited thereto. In one embodiment of the present invention, BCECF-AM was used as a dye showing fluorescence in the cells.

Conversion into a fluorescent substance in the cell may be performed by an intracellular enzyme, for example, the enzyme may be an esterase.

When BCECF-AM is treated to the target cell to be measured, BCECF-AM penetrates the cell membrane and flows into the cell by diffusion, and is separated into BCECF and AM by intracellular enzymes, for example, esterases, and intracellular pH. Depending on the BCECF, the amount of fluorescence is changed, and the amount of fluorescence is proportional to the pH, so that the intracellular pH can be measured.

According to the present invention, when a certain amount of BCECF-AM is treated, it is possible to improve the problem of the conventional intracellular pH measurement method in which the fluorescence value of BCECF is differently measured according to the number of cells even at the same pH.

In one embodiment of the present invention, the concentration of the dye can be treated to the cells to increase in proportion to the number of cells. The inventors of the present application confirmed that by adjusting the concentration of the dye to be treated in the cell to be measured in pH in proportion to the number of cells, it is possible to measure the exact pH regardless of the number of cells to which the dye is treated.

Specifically, the concentration (quantity) of the dye to be treated for intracellular pH measurement can be determined according to the following equation.

Dye concentration to be applied (amount) = Reference dye concentration (amount) X (number of cells to be measured/number of reference cells)

In the above formula, the reference cell number means the number of cells counted based on the case where the cells are seeded to be 60 to 70% of the dish or plate immediately before the pH measurement.

It is preferable to set the reference dye concentration (quantity) to a concentration at which the dye is not saturated. The reference dye concentration (quantity) can be derived by measuring the fluorescence value through a flow cytometry based on an arbitrary amount, for example, BCECF-AM 0.5 mg/cell culture medium L, and the fluorescence value when treated with a plurality of concentrations The amount of dye is increased when the indicated peak is shifted to the left, and the amount of dye is decreased when the peak indicating fluorescence is shifted to the right, and when the peak is visually identified, it is located at the center of the graph y-axis. The amount can be a reference dye concentration (quantity).

Thereafter, the number of cells to be measured is counted, and the ratio of the number of cells to be measured to the number of reference cells is determined, and then multiplied by the reference dye concentration (amount) to determine the dye concentration (amount) to be applied.

In one embodiment of the present invention, the dye may be treated at a concentration calculated in proportion to the number of cells counted, based on a concentration of 1 mg/ml per 1.5 X 10 ⁵ to 6.0 X 10 ⁵ cells. When measuring pH in MDA-MB-231 cells, BCECF-AM concentration is 0.5 mg/ml when the number of cells to be measured is 3.0 X 10 ⁵ , based on the concentration of BCECF-AM per cell number of 6.0 X 10 ⁵ cells. It was confirmed that the AM treatment can accurately measure the pH regardless of the number of cells.

In another embodiment of the present invention, when measuring the pH in SNU81 cells, based on the concentration of BCECF-AM per 1.5 X 10 ⁵ cells is 1 mg/ml, when the number of cells to be measured is 3 X 10 ⁵ , 2 mg/ml When BCECF-AM treatment was performed at a concentration, it was confirmed that an accurate pH can be measured regardless of the number of cells.

The dye may be treated with cells at a concentration calculated in proportion to the number of cells, for example, in direct proportion. In one embodiment according to the present invention, when the number of cells is doubled based on the dye concentration of 1 mg/ml per 1.5 X 10 ⁵ to 6.0 X 10 ⁵ cells, the concentration of the dye to be treated was also increased to 2 times, As a result, it was confirmed that an accurate pH can be measured regardless of the number of cells to which the dye is treated.

The cells may be, for example, breast cancer cell line MDA-MB-231 cells or Korean colon cancer cell line SNU81, but are not limited thereto.

[Example]

Hereinafter, the present invention will be described in more detail by specific examples. However, the present invention is not limited by the following examples, and it will be apparent to those skilled in the art that various modifications or modifications can be made within the spirit and scope of the present invention.

Example 1: pH measurement in breast cancer cell line

BCECF-AM (2',7'-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein, Acetoxymethyl Ester) is the most widely used intracellular pH probe. BCECF-AM can be easily penetrated into cells, and after penetration into cells, AM is cut by the action of ester hydrolase in the cytoplasm, and BCECF has 4 to 5 negative charges at neutral pH and is highly water-soluble and difficult to be discharged out of the cell. . Based on this principle, BCECF is a pH-dependent dye that exhibits green fluorescence. The excitation and emission wavelengths were 535 nm and 490 nm, respectively.

BCECF-AM showed the difference in the saturation degree according to the number of cells, and a preliminary experiment was conducted to select a concentration of 0.5 mg/L in MDA-MB-231 cells and 0.125 mg/L in SNU-81 cells, and the treatment time was 1 It was time.

When the pH is measured for each different number of cell groups, a difference in the amount of absorption of BCECF-AM per cell occurs, so the pH measurement value according to the fluorescence density is not accurate.

Therefore, in order to match the uptake of BCECF-AM in the cells, the groups were divided. In order to evaluate the accuracy of BCECF-AM according to the number of cells, a method of comparing fluorescence expression of BCECF-AM after dispensing with various cell numbers and a method of comparing fluorescence expression while controlling BCECF-AM concentration according to the number of cells were used. . One group treated the same amount of BCECF-AM regardless of the number of cells of 300,000, 600,000, 1.2, 1.8, and 2.4 million cells, and the other group treated the concentration of BCECF-AM differently for each cell, and later drugs Differences in BCECF-AM uptake by cell number changes were compared according to treatment.

MDA-MB-231 cells growing in RPMI1640 growth medium mixed with 10% FBS were washed with PBS (Phosphate buffered saline, 2.7 mM KCl, 1.5 mM KH2PO4, 1.8 mM Na ₂ HPO ₄ , pH 7.4) and then trypsinized. After harvesting the cells by unicellularization, counting the number of viable cells using a hematocytometer, a group of 300,000, 600,000, 1.2 million, 1.8 million, and 2.4 million cells were used for cell culture in the amount of 2 ml. In 24 well Petri dishes, FBS was added to RPMI1640 maintenance medium, and the other groups dispensed 300,000, 600,000, 1.2, 1.8, and 2.4 million cells in an amount of 600,000 cells/ml. At this time, the concentration of BCECF-AM was treated with 0.5 mg/medium L at 37° C. for 1 hour. After that, the washing process was performed twice using PBS and dispensed with 1 ml of PBS per 600,000 cells. All of these processes were maintained at 4°C (on ice). The prepared sample was measured for fluorescence using flow cytometry (BD Accuri ^TM C6 plus) to check the intracellular pH.

Live cells are selected based on the size and shape of the cells using the FSC/SSC graph using the BD Accuri ^TM C6 plus software to obtain fluorescence values through the FL-1 channel. The median was compared in the histogram of the FL-1 channel.

Referring to the result of FIG. 1 as an example, when BCECF-AM is treated with 2 ml regardless of the number of cells, despite the cells having the same pH, flow cytometry results (A02) The cell group graph is not consistently collected and the number of cells As it decreases, you can see the phenomenon of biasing to the right (purple right graph in 300,000 cells). In addition, if you look at the mean or median FL1-A values in the table, you can see that they appear the same as the black pattern of the line graph. This means that as the number of cells decreases, the amount of BCECF-AM per cell increases. However, when the amount of BCECF-AM was differently processed according to the number of cells, it was confirmed that the graphs of all groups overlapped in the flow cytometry result (B01). Also in the table, it can be seen that the mean and median FL1-A values are almost the same, so they are kept constant like the red pattern of the line graph.

The result of FIG. 4 is a graph obtained by performing the above experiment method independently three times, and it can be seen that even if the experiment is repeatedly performed, stable results can be obtained without significant difference in values.

Example 2: pH measurement in colorectal cancer cell line

For SNU-81 cells, SNU-81 cells grown in RPMI1640 growth medium containing 10% FBS are washed with PBS (2.7 mM KCl, 1.5 mM KH ₂ PO ₄ , 1.8 mM Na ₂ HPO ₄ , pH 7.4). After the cells were harvested by unicellularization by trypsin treatment, the number of viable cells was counted using a hematocytometer. Dispense into RPMI1640 maintenance medium without FBS as a 60 mm or 100 mm petri dish for culture, and in the other groups, 300,000, 600,000, 1,200,000, and 2,400,000 cells of 600,000 cells/ml of medium Dispense by volume. At this time, the concentration of BCECF-AM is treated with 0.5 mg/medium L at 37°C for 1 hour. Thereafter, the washing process was performed twice using PBS, and after dispensing with 1 ml of PBS per 600,000 cells, the analysis method was performed in the same manner as in Example 1.

The results were also the same as in Example 1. When the amount of BCECF-AM was treated differently according to the number of cells, it was confirmed that the graphs of all groups overlapped in the flow cytometry result (B01). Also in the table, it can be seen that the mean and median FL1-A values are almost the same, so they are kept constant like the red pattern of the line graph.

The result of FIG. 8 is a graph obtained by performing the above experiment method independently three times, and it can be seen that even if the experiment is repeatedly performed, stable results can be obtained without a significant difference in values.

The specific parts of the present invention have been described in detail above. For those skilled in the art, this specific technique is only a preferred embodiment, and the scope of the present invention is not limited thereby. It will be obvious. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (5)

As a method for improving the accuracy of intracellular pH measurement,
Comprising the step of treating a dye that exhibits fluorescence in the cell to the cell to be measured pH,
The concentration of the dye is characterized in that the number of cells to be measured for pH is adjusted based on the number of cells.
The method of claim 1, wherein the dye that exhibits fluorescence in the cell is BCECF-AM.
According to claim 1, The concentration of the dye is determined according to the formula of the reference dye concentration (quantity) X (the number of cells to be measured / the number of reference cells), the reference dye concentration (quantity) and the reference cell number is the fluorescence value A method characterized in that the ratio between the average value and the median value of the peaks shown is about 1 and the number of cells.
The method according to claim 1, wherein the dye is treated at a concentration calculated in proportion to the number of cells to be measured, based on a concentration of 1 mg/ml per 1.5 X 10 ⁵ to 6.0 X 10 ⁵ cells. .
The method according to claim 4, wherein the dye is treated at a concentration calculated in direct proportion to the number of cells to be measured, based on a concentration of 1 mg/ml per cell number of 1.5 X 10 ⁵ to 6.0 X 10 ⁵ cells.

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