Method for measuring lysosomal activity

Abstract

To provide a method with which it is possible to simply measure lysosomal activity of cells in a sample.　The method for measuring intracellular lysosomal activity in vitro comprises the following steps (1)-(4): (1) A step for adding a fluorescent substance-binding protein to a medium containing cells and culturing the cells; (2) a step for recovering cultured cells after step (1); (3) a step for adding a composition for cell lysis to the cultured cells recovered in step (2) to obtain a cell lysate fraction; and (4) a step for measuring the fluorescence intensity of the fluorescent substance-binding protein in the cell lysate fraction obtained in step (3).

Description

Methods for measuring lysosomal activity

[0001] The present invention relates to a method for measuring lysosomal activity.

[0002] Lysosomes are intracellular organelles with an acidic membrane-enclosed interior and are characterized by their enzymatic activity, which degrades various biopolymers through the actions of proteases, glycosidases, lipases, nucleases, etc. Because lysosomes are the end point of multiple pathways, including endocytosis and autophagy, they are known to play a wide range of physiological roles both directly and indirectly.

[0003] Autophagy is one of the lysosome-related protein degradation pathways. Autophagy is a bulk degradation system commonly found in eukaryotic cells, enabling cellular homeostasis by constantly replacing cytoplasmic components. When autophagy is induced in response to various stimuli, autophagosomes, double-membrane vesicles, fuse with lysosomes to form autolysosomes, where unnecessary proteins are degraded. Abnormalities in the autolysosomal proteolytic function are known to contribute to various pathologies, including cancer, neurodegenerative diseases, cardiovascular diseases, pulmonary diseases, and infectious diseases, as well as to biological phenomena such as aging and motor function. Autophagy has been reported to play an important role in hearing and cell morphology.

[0004] Although various approaches have been used to measure intracellular lysosomal activity for a long time, no unified method has been established (Patent Document 1). Classical methods for detecting lysosomal protease activity include cathepsin maturation assays using a pulse-chase method with radioisotopes and immunoblotting with anti-cathepsin antibodies. Although these methods are widely used, the former requires the use of radioisotopes, while the latter has problems with measurement errors due to cell density, probe concentration, and uptake rate. Furthermore, a method using LysoTracker Dye, which targets the acidic contents of lysosomes, is known, but has not been used for quantitative measurement (Non-Patent Document 1). Reported methods for measuring lysosomal activity include a quantitative measurement method incorporating a fluorescent probe into a plasmid and a plasmid-based lysosomal activity measurement method using a flow cytometer, but these methods are not easy to use (Patent Document 1, Non-Patent Document 2). Both of these methods require complex procedures, and unless performed by an experienced technician, results can vary from measurement to measurement, making it difficult to obtain reliable results.

[0005] A method for measuring lysosomal activity has been developed in which DQ™-BSA, a bovine serum albumin conjugated to the fluorescent dye BODIPY, is degraded within cells and the emitted fluorescence is measured under a microscope (Non-Patent Document 3). Because this method requires measurement under a microscope, obtaining highly accurate and reliable results requires not only sample handling but also image manipulation, obtaining reliable fluorescence intensity data, removing background, and studying ways to improve reliability so that the method can withstand repeated use, making it not necessarily a simple measurement method.

[0006] Furthermore, all of the above-mentioned conventional methods are based on the assumption that they will be used in cultured cells. While analyses using cultured cells are powerful tools for selective visualization, such as investigating the intracellular localization of target substances, microscopic observation requires specialized equipment and complicated procedures. Furthermore, because analysis is limited to a limited field of view, verification is required to ensure accurate representation of the entire sample. Furthermore, it is necessary to consider factors such as the viability of the target cells, reproducibility between batches, and the influence of the culture medium.

[0007] International Publication No. 2011 / 019082

[0008] Cytometry Part A, 85A; 169-178, 2014Sci. Rep. 9; 11635, 2019Bio Protoc. 7(19); e2571, 2017

[0009] An object of the present invention is to provide a method that can easily measure the lysosomal activity of cells in a sample without observing the cells using a microscope.

[0010] In light of the above-mentioned circumstances, the inventors conducted extensive research and found that by adding a fluorescent substance-binding protein, recovering cultured cells from a cell culture vessel, lysing the cells with a cell lysis solution, and measuring the fluorescence intensity, it is possible to easily measure lysosomal activity in a stable and highly accurate manner without being affected by interfering substances and without relying on cell observation.

[0011] That is, the present invention provides the following: [1] A method for measuring intracellular lysosomal activity in vitro, the method comprising the following steps (1) to (4): (1) culturing the cells in a medium containing a fluorescent substance-binding protein; (2) recovering the cultured cells after step (1); (3) adding a cell lysis composition to the cultured cells recovered in step (2) to obtain a cell lysate fraction; and (4) measuring the fluorescence intensity of the cell lysate fraction obtained in step (3). [2] The method according to [1], further comprising steps (5) and (6) after step (4): (5) measuring the protein concentration of the cell lysate fraction; and (6) calculating the value (RFU (Relative Fluorescence Units)) obtained by dividing the fluorescence intensity of step (4) by the protein concentration measured in step (5). [3] The method according to [1] or [2], wherein the fluorescent substance is at least one selected from the group consisting of FITC, Cy3, Cy5, and BODIPY. [4] The method according to any one of [1] to [3], wherein the fluorescent substance is BODIPY and the protein is bovine serum albumin (BSA). [5] The method according to any one of [1] to [4], wherein the cell lysis composition is at least one selected from the group consisting of CHAPS lysis buffer, HBST, cell lysis buffer, Buffer A, M-PER buffer, cell lysis buffer M, and RIPA buffer. [6] The method according to any one of [1] to [5], wherein the cells are at least one of HeLa cells, HEK293 cells, HEI-OC1 cells, and HUVEC. [7] A method for assisting in the evaluation of the effectiveness of a disease treatment in a subject, comprising measuring intracellular lysosomal activity in a sample removed from the subject by the method of claim 1. [8] A kit for measuring intracellular lysosomal activity in vitro, comprising: a first measurement reagent solution containing a fluorescent substance-binding protein; and a second measurement reagent solution containing a cell lysis composition. [9] The kit according to [8], further comprising a third measurement reagent solution for quantifying protein concentration. This specification incorporates the disclosure of Japanese Patent Application No. 2023-182873, from which the present application claims priority.

[0012] According to the present invention, it is possible to provide a method that can simply measure the lysosomal activity of cells in a sample without observing the cells using a microscope.

[0013] 1 is a graph showing the results of measuring lysosomal activity in HEK293 cells in the presence of BafA1 by co-staining with DQ Green BSA and DAPI as a comparative example. The test was performed three times on different days, with N=4 for each test. The error bars in the graph indicate standard error. In Example 1, this graph shows the results of measuring lysosomal activity in HEK293 cells in the presence of BafA1 by staining with DQ Green BSA and lysing cells with a cell lysate. The test was performed three times on different days, with N=3 for each test. The error bars in the graph indicate standard error. In Example 2, this graph shows the results of measuring lysosomal activity in HeLa cells, HEI-OC1 cells, and HUVEC in the presence of various concentrations of BafA1 by staining with DQ Green BSA and lysing cells with a cell lysate. A shows the lysosomal activity of HeLa cells, B shows HE1-OC1, and C shows HUVEC. The test was performed with N=3. The error bars in the figure indicate standard error. In Example 3, this graph shows the results of measuring lysosomal activity in HEK293 cells in the presence of BafA1 or various concentrations of CQ, by staining with DQ Green BSA and lysing the cells with a cell lysis solution. N=3 was performed for each run. The error bars in the figure indicate standard error. In Example 4, this graph shows the results of measuring lysosomal activity in HEK293 cells, after lysing the cells using CHAPS buffer and Triton X-100 buffer. The test was performed with N=3. The error bars in the figure indicate standard error. 1 is a graph showing the results of measuring lysosomal activity in HEK293 cells in the presence of BafA1 by staining with DQ ovalbumin and lysing the cells with a cell lysis solution, as Example 5. N=3 was used for the experiment. Error bars in the figure indicate standard error.

[0014] 1. Structure and Definition <Sample> As used herein, the term "sample" is not particularly limited as long as it contains cells that can be used to measure lysosomal activity, and encompasses all organs, tissues, cells, and the like. Examples include organs, tissues, and cells derived from vertebrates such as mammals, birds, reptiles, amphibians, and fish. Mammals are preferred, and humans or laboratory animals (e.g., mice, rats, hamsters, and rabbits) are more preferred. The term "cells" as used herein is not particularly limited as long as they contain proteins. They may be in any form, such as cell lines, primary cultured cells, and tissue-derived sections, and may be derived from any specimen. Cultured cells are particularly suitable. The term "tissue" as used herein is not particularly limited, and may include, for example, tissues cultured under special conditions, such as cancer tissue and ischemic tissue. Samples may be collected from tissues or cells, or from the environment of the tissues or cells. In some examples, the sample may be tissue biopsy material, blood, plasma, extracellular fluid, cultured cells, culture medium, discarded tissue, plant material, synthetic proteins, archaea, bacteria, mycelial tissue, or protozoa. Examples of cultured cells or primary cultured cells include, but are not limited to, human cervical cancer cells (HeLa cells), human embryonic kidney cells (HEK293 cells), mouse cochlear cells (HEI-OC1 cells), and human umbilical vein endothelial cells (HUVEC).

[0015] <Fluorescent substance-binding protein> In this specification, the term "fluorescent substance-binding protein" refers to a protein bound to a fluorescent substance, also known as a "self-quenching dye," which is configured to emit fluorescence upon being taken up into a lysosome and hydrolyzing the protein portion. The protein constituting the "fluorescent substance-binding protein" is not particularly limited, but for example, bovine serum albumin (BSA) can be preferably used. In this specification, the term "fluorescent substance" refers to a substance such as an organic compound or protein that emits fluorescence of a specific fluorescent wavelength when irradiated with light of a specific excitation wavelength. Known organic compounds include, for example, FITC (Molecular Probes), Texas Red (Molecular Probes), Cy3 and Cy5 (General Electric Healthcare), 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY (trademark)), boron dipyrromethene dyes (BODIPY dyes) such as BDP-FL, BIP, CF-MONO, CF-BI, and BDPFL NHS-Ester. Known proteins include, for example, GFP, CFP, RFP, and YFP. Preferably, BODIPY FL and BODIPY TR can be used.

[0016] As the "protein bound to a fluorescent substance," for example, a commercially available product can be used. Known examples of such a commercially available product include DQ-BSA (manufactured by Thermo Fisher), in which BODIPY is bound to BSA.

[0017] <Cell Lysis Composition> As used herein, the term "cell lysis composition" refers to any solution composition generally used in cell lysis or protein solubilization. For example, known solutions such as CHAPS lysis buffer, RIPA buffer, HBST, cell lysis buffer, tissue lysis buffer, and Buffer A can be used. These are also commonly referred to as "cell lysis reagents" or "cell lysis solutions." Those skilled in the art can select and use any of these solutions appropriately. The cell lysis composition may contain a buffer. Buffers that have a buffering effect near weak alkalinity, such as PBS, Tris, and HEPES, can be used.

[0018] The cell lysis composition may contain a surfactant such as CHAPS, SDS, NP40, or Triton-X to solubilize proteins. It may also contain urea, which also has a solubilizing effect, and can be selected appropriately for use.

[0019] A protease inhibitor may be contained to prevent protein degradation by intracellular proteases. The protease inhibitor may be appropriately selected from commonly used agents such as PMSF (phenylmethylsulfonyl fluoride), aprotinin, leupeptin, pepstatin, sodium fluoride, and sodium orthovanadate. Furthermore, a divalent metal ion may be used as a sequestering agent (chelating agent) to inhibit proteases. Examples of chelating agents that can be used include EDTA and EGTA. Furthermore, a phosphatase inhibitor such as sodium fluoride, sodium orthovanadate, sodium pyrophosphate, and β-glycerophosphate may be used to maintain the phosphorylated state of proteins.

[0020] To cleave disulfide bonds in proteins, reducing agents such as DTT (dithiothreitol) and BME (β-mercaptoethanol) may be used.

[0021] Those skilled in the art can appropriately select and prepare appropriate concentrations of these additives contained in the cell lysis composition. For example, RIPA buffer may contain 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100 or NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, and 10 mM NaF. Depending on the application, these compositions can be appropriately modified, or additives such as protease inhibitors can be added. Those skilled in the art can appropriately modify and prepare these compositions depending on the purpose of the experiment. In addition, commercially available products such as M-PER buffer (manufactured by Thermo Fisher) and Cell Lysis Buffer M (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) may also be used. Preferably, RIPA buffer can be used.

[0022] As used herein, the term "kit" refers to an assembly of multiple elements used to carry out a single method. As used herein, a "reagent" refers to a single element. Here, a test solution contained in a single container constitutes one element.

[0023] 2. Method for Measuring Intracellular Lysosomal Activity A first embodiment of the present invention is a method for measuring intracellular lysosomal activity. The method of this embodiment is a method for measuring intracellular lysosomal activity in vitro, and is characterized by comprising the following steps (1) to (4): (1) culturing the cells in a medium containing a fluorescent substance-binding protein; (2) recovering the cultured cells after step (1); (3) adding a cell lysis composition to the cultured cells recovered in step (2) to obtain a cell lysate fraction; and (4) measuring the fluorescence intensity of the fluorescent substance-binding protein in the cell lysate fraction obtained in step (3).

[0024] The method of the present embodiment includes a step of lysing cells when measuring intracellular lysosomal activity, thereby enabling quantitative measurement of lysosomal enzyme activity without cumbersome procedures such as image analysis, etc. Therefore, lysosomal activity can be measured simply and accurately, regardless of the skill level of the operator.

[0025] <Step (1): Culturing Step> Hereinafter, each step of the method of this embodiment will be described. Hereinafter, the method will be described along the steps of measuring lysosomal activity using HEK293 cells as an example, but is not intended to limit the scope of the present invention.

[0026] Step (1) of the method of this embodiment is a step of adding a fluorescent substance-binding protein to a medium containing cells and culturing the cells. Prior to step (1), the cells are preferably pre-cultured in a cell culture dish or the like. The pre-culture is carried out, for example, using DMEM medium at 37°C for 24 hours or more.

[0027] The medium after the preliminary culture is replaced with a medium containing the fluorescent substance-binding protein, and the cell culture is continued. The concentration of the fluorescent substance-binding protein is not particularly limited, but can be about 1 to 100 μg / mL, 5 to 50 μg / mL, and particularly about 10 μg / mL. The culture temperature is preferably 30 to 40°C, and particularly 32 to 38°C. The culture time is preferably 10 minutes to 24 hours, and particularly 30 minutes to 8 hours. The culture temperature and culture time can be adjusted appropriately depending on the type and properties of the cells used.

[0028] <Step (2): Recovery Step> Step (2) of the method of this embodiment is a step of recovering the cultured cells after step (1). Depending on the type of cell, a protease such as trypsin may be used to recover the cells. For example, the cultured cells can be recovered by centrifugation at 4°C and 1000 x g (approximately 2000 rpm) for 5 minutes. At this point, the cells may be washed with ice-cold PBS or the number of cells may be counted.

[0029] <Step (3): Cell Lysis Step> Step (3) of the method of this embodiment is a step of adding a cell lysis composition to the cultured cells recovered in step (2) to obtain a cell lysate fraction. Here, a step using RIPA buffer as the cell lysis composition will be described as an example.

[0030] Chilled RIPA buffer is added to the cells collected in step (2). Preferably, the RIPA buffer contains a protease inhibitor. It is preferable to add 100 μL of RIPA buffer per 10 cells collected. However, depending on the cell type and conditions, the volume of RIPA buffer can be increased or decreased as needed, for example, when a higher protein concentration is expected. The amount of RIPA buffer used can be appropriately selected by those skilled in the art. The RIPA buffer containing the cells is then stored on ice for 30 minutes while repeatedly vortexing and mixing, and can be used for measuring lysosomal activity.

[0031] When the cells to be measured are derived from tissue, the tissue can be dissected and, if necessary, washed with chilled PBS to remove blood. After cutting the tissue into small pieces while chilling on ice, the tissue is placed in a homogenizer and RIPA buffer containing a protease inhibitor is added. The amount of RIPA buffer used may be, for example, 500 μL per approximately 10 mg of tissue. The RIPA buffer containing the tissue can be completely homogenized in a homogenizer and left on ice for 30 minutes before use in measuring lysosomal activity. If necessary, a sonication step can be performed. In the sonication step, the sample is sonicated to further disrupt the cells or tissue and cleave the DNA. The sonication time can be adjusted appropriately depending on the type of sample. For example, while the sample is cooled on ice, cell lysis can be carried out at an output of 180 watts for 1 minute, and tissue lysis can be carried out for 2-5 minutes (repeated as one cycle of 10 seconds of ultrasound and 10 seconds of rest).

[0032] <Step (4): Fluorescence Measurement Step> Step (4) of the method of this embodiment is a step of measuring the fluorescence intensity of the fluorescent substance-binding protein in the cell lysate fraction obtained in step (3). The method for measuring the fluorescence intensity of the cell lysate fraction is not particularly limited, and existing methods such as a plate reader or flow cytometry can be used as appropriate. For simple and rapid measurement, it is preferable to measure the fluorescence intensity using a plate reader. An example of a step using a plate reader will be described below.

[0033] Lysosomal activity can be measured by adding the cell lysate fraction extracted with the cell lysis composition to a black 96-well plate for luminescence / fluorescence analysis and measuring the fluorescence intensity using a plate reader (e.g., manufactured by BMG Labtech) (when using DQ-BSA, the excitation wavelength is 485 nm and the fluorescence wavelength is 520 nm). Measurements may be performed once for each sample, or values ​​obtained by multiple measurements may be used. For more accurate lysosomal activity measurements, multiple measurements are preferred. The measured fluorescence intensity can be corrected using protein concentration or the like to enable more accurate measurement of lysosomal activity. For example, the fluorescence intensity can be used by subtracting the background fluorescence intensity of the RIPA buffer from the fluorescence intensity.

[0034] <Step (5): Protein Concentration Measurement Step> The method of this embodiment may further include steps (5) and (6) after step (4). Step (5) of the method of this embodiment is a step of measuring the protein concentration of the cell lysate fraction. The method for measuring the protein concentration is not particularly limited, and any known method can be used. For example, the method can be performed by a general method using the BCA method. The BCA method can be used to perform quantification using the Pierce BCA Protein Assay Kit (manufactured by Thermo Fisher Scientific).

[0035] <Step (6): RFU Calculation Step> Step (6) of the method of this embodiment is a step of calculating the value (RFU (Relative Fluorescence Units)) obtained by dividing the fluorescence intensity of step (4) by the protein concentration measured in step (5). By expressing lysosomal activity in RFU, it becomes possible to more accurately evaluate the presence or absence of a change in lysosomal activity, for example, when evaluating the effect of a drug or the like that acts on lysosomes.

[0036] <Application Examples> The method of this embodiment, having the above-described configuration and steps, can easily measure intracellular lysosomal activity, and is expected to contribute to research on various diseases, including lysosomal storage disorders and cancers and neurodegenerative diseases in which autophagy is thought to be involved, as well as aging and motor function. Furthermore, the method of this embodiment can be effectively used to evaluate the drug concentration dependency of diseases, screen therapeutic drugs, and as an index for confirming the validity of treatment strategies for various diseases. In particular, it can be used as a method for monitoring therapeutic effects to evaluate the effectiveness of disease treatment.

[0037] An example application of the method of this embodiment is a method for screening for drugs capable of recovering from lysosomal damage. The inventors found that the intracellular lysosomal activity measured by the method of this embodiment decreases in a lysosomal-damaging agent concentration-dependent manner when cells are coexistent with the lysosomal-damaging agent. This demonstrates that the method of this embodiment can quantitatively evaluate both the degree of lysosomal damage and the degree of recovery. This screening method can include, for example, the following steps (a) to (d): (a) culturing cells in a medium containing a candidate drug, a lysosomal-damaging agent, and a protein bound to a fluorescent substance; (b) recovering the cultured cells after step (a); (c) adding a cell lysis composition to the cultured cells recovered in step (b) to obtain a cell lysate fraction; and (d) measuring the fluorescence intensity of the fluorescent substance-bound protein in the cell lysate fraction obtained in step (c). By confirming in steps (b) to (d) the extent to which the candidate drug restores the lysosomal activity reduced by the action of the lysosomal damaging agent in step (a), it is possible to easily confirm the ability of the candidate drug to restore lysosomal activity.

[0038] Examples of "lysosome-damaging agents" that can be used include known lysosome-damaging agents such as bafilomycin A1 (hereinafter sometimes referred to as "BafA1") and chloroquine (hereinafter sometimes referred to as "CQ"). In addition, any substance having lysosome-damaging activity can be used, such as hydroxychloroquine, which has a structure similar to chloroquine, bisbenzylisoquinoline alkaloids such as azithromycin, and macrolide antibiotics such as tetrandrine.

[0039] 3. Method for assisting in the evaluation of the effectiveness of disease treatment A second embodiment of the present invention is a method for assisting in the evaluation of the effectiveness of disease treatment in a subject. The method of this embodiment is characterized by comprising measuring intracellular lysosomal activity in a sample extracted from a subject by the method of the first embodiment. In the method of this embodiment, the reagents, instruments, samples, conditions, etc. used are the same as those of the first embodiment unless otherwise specified or unless otherwise contradictory.

[0040] Intracellular lysosomal activity can be used to evaluate the effectiveness of treatments for various diseases as an indicator of the appropriateness of treatment strategies. Because numerous enzymes and associated proteins reside in lysosomes, functional abnormalities, such as decreased enzyme activity, are known to cause lysosomal diseases such as sphingolipidoses, mucopolysaccharidoses, oligosaccharide storage diseases, glycogen storage diseases, neutral lipid storage diseases, and I-cell disease (mucolipidosis II). These represent more than 40 types of congenital metabolic disorders that cause various symptoms and are designated as "designated intractable diseases" in Japan. The method of this embodiment can be used to obtain an indicator of the appropriateness of treatment strategies for diseases such as lysosomal diseases, which are known to be highly associated with cellular lysosomal activity. For example, in vitro lysosomal activity measurements of samples extracted from a subject and comparison with control values ​​can be used as an indicator for determining the dosage of a therapeutic drug.

[0041] For example, a predetermined dose of a therapeutic drug is administered to a subject suffering from a disease known to be highly associated with lysosomal activity, and the measured value of the patient's intracellular lysosomal activity is compared with a preset cutoff value or a control value before administration of the therapeutic drug, and the therapeutic drug dose can be increased or decreased. For example, if lysosomal activity is low compared with a preset cutoff value or a control value before administration of the therapeutic drug, the therapeutic drug dose can be increased or decreased from the original therapeutic drug dose and applied to the subject. For example, if lysosomal activity is high compared with a preset cutoff value or a control value before administration of the therapeutic drug, the therapeutic drug dose can be decreased or applied to the subject.

[0042] 4. Lysosomal Activity Measurement Kit The third embodiment of the present invention is a kit for measuring intracellular lysosomal activity in vitro. The kit of this embodiment is characterized by comprising: a first measurement reagent solution containing a fluorescent substance-binding protein; and a second measurement reagent solution containing a cell lysis composition. More specifically, the kit of this embodiment is a kit used in the method of the first embodiment. In the kit of this embodiment, the reagents, instruments, samples, and conditions of use, etc. used are the same as those of the first embodiment unless otherwise specified or unless otherwise contradicted.

[0043] The kit of this embodiment has at least two components: a first measurement reagent solution containing a fluorescent substance-binding protein and a second measurement reagent solution containing a cell lysis composition. The first measurement reagent solution can be a culture medium containing the fluorescent substance-binding protein. Alternatively, the first measurement reagent solution can be a buffer solution containing a high concentration of the fluorescent substance-binding protein. The second measurement reagent solution can be a cell lysis composition (e.g., RIPA buffer) or a 5x solution of the cell lysis composition (for dilution before use).

[0044] The kit of this embodiment may include a composition for quantifying protein concentration as the third measurement reagent solution. Any known composition for quantifying protein concentration may be used as the composition for quantifying protein concentration. For example, when quantifying protein by the BCA method, the kit may include a bicinchoninic acid solution and a copper sulfate solution.

[0045] In addition, the kit of this embodiment may include a cell culture dish, a black microplate for fluorescence measurement, instructions for use (package insert), and the like, as needed.

[0046] [Comparative Example] Measurement of intracellular lysosomal activity by co-staining with DQ Green BSA and DAPI. HEK293 cells were cultured in 8-chamber slides at 2 × 10 ４ The cells were diluted with DMEM medium to give 100 cells / well and seeded. The seeded cells were incubated at 37°C, 5% CO ２After 24 hours of culture under these conditions, the culture supernatant was removed, and DMEM medium containing 0 (control), 200 nM Bafilomycin A1 (BafA1, Bioaustralis), and 10 μg / mL DQ Green BSA was added and incubated at 37°C for 6 hours. The supernatant was removed, and the cells were fixed with 1% paraformaldehyde and stained with DAPI. Images were taken with a fluorescence microscope (KEYENCE) (exposure time: GFP 4 seconds, DAPI 1 / 200 seconds), and the green fluorescence area was calculated using Image J with a standard threshold of 19-255. The value was divided by the number of DAPI-stained cell nuclei and graphed (Figure 1). Four samples were sampled per group. The test was performed three times on different days. The error bars in the figure indicate standard errors.

[0047] The results of three tests are shown in Figure 1. There was a large discrepancy in the measured values ​​between the days of testing, and it was not possible to clearly detect a decrease in lysosomal activity due to the addition of BafA1. This was thought to be because the field of view within the well was manually selected when capturing images using a fluorescence microscope, resulting in large differences between operators and samples, and insufficient accuracy and reproducibility.

[0048] Example 1 Measurement of intracellular lysosomal activity using DQ Green BSA and cell lysate HEK293 cells were plated in a 6-well plate at 3 × 10 ５ The cells were diluted with DMEM medium to give 100 cells / well and seeded. The seeded cells were incubated at 37°C, 5% CO ２ After culturing for 24 hours under these conditions, the culture supernatant was removed, and DMEM medium containing 0 (control), 100 nM BafA1, and 10 μg / mL DQ Green BSA was added, followed by incubation at 37°C for 6 hours. The supernatant was removed, and the cells were collected. RIPA buffer (50 mM Tris-HCl, 0.1% SDS, 0.5% DOC, 1% NP-40, 150 mM NaCl (pH 8.0)) was added, and the mixture was cooled on ice for 10 minutes. This allowed intracellular proteins to be extracted, and an extract was obtained.

[0049] The extract was dispensed into a black 96-well plate for luminescence and fluorescence analysis at 150 μL / well, and the fluorescence intensity (excitation wavelength: 485 nm, fluorescence wavelength: 520 nm) was measured using a plate reader (BMG Labtech). The protein content of the extract was also quantified using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific).

[0050] The background fluorescence intensity of the RIPA buffer was subtracted from the fluorescence intensity of the sample at each BafA1 concentration, and the result was divided by the protein concentration in the extract (RFU). N=3 samples were collected for each group. The test was performed three times on different days.

[0051] The measurement results of three tests are shown in Figure 2. The error bars in the figure indicate standard error. The error between days was small, indicating that highly reproducible results were obtained compared to Comparative Example 1. It was also shown that the reduction in lysosomal activity caused by BafA1 could be reliably observed. This is thought to be because, unlike measurements using a fluorescence microscope, signals can be measured in bulk without the need for manual field selection, etc.

[0052] [Example 2] BafA1 concentration dependence of lysosomal activity measurements HeLa cells, HEI-OC1 cells, and HUVECs were used to confirm the BafA1 concentration dependence of lysosomal activity measurements. HeLa cells, HEI-OC1 cells, and HUVECs were cultured in a 6-well plate at 3 × 10 ５ The seeded cells were diluted with DMEM medium to give 100 cells / well. The seeded HeLa cells and HUVECs were incubated at 37°C and 5% CO ２ , HEI-OC1 cells were incubated at 33°C and 10% CO ２After 24 hours of culture under these conditions, the culture supernatant was removed, and DMEM medium containing 0, 1, 10, or 100 nM BafA1 was added to HeLa cells and HEI-OC1 cells, and 0, 0.1, 1, or 10 nM BafA1 was added to HUVECs. The HeLa cells and HUVECs were incubated at 37°C for 6 hours, and the HEI-OC1 cells were incubated at 33°C for 1 hour. Subsequently, extract preparation, fluorescence measurement, background measurement, and protein concentration measurement were performed as in Example 1. The background was subtracted from the fluorescence measurement value of each sample and the result was divided by the protein concentration to calculate RFU. The RFU value of the sample without BafA1 was set to 100%, and the relative lysosomal activity (%) was calculated from the RFU value of each sample with BafA1 added, and the results were graphed (Figure 3). N=3 samples were used for each group.

[0053] The BafA1 concentration dependence of lysosomal activity in each cell type is shown in Figure 3. Figure 3A shows the results for HeLa cells, Figure 3B shows the results for HEI-OC1 cells, and Figure 3C shows the results for HUVEC. Error bars in the figures indicate standard error. As shown in the figures, it was confirmed that lysosomal activity decreased in a BafA1 concentration-dependent manner in all of HeLa cells, HEI-OC1 cells, and HUVEC. It was demonstrated that the method of the present invention can be used to detect drug concentration-dependent impairment of lysosomal activity and differences in drug sensitivity among cells.

[0054] [Example 3] Concentration dependence of lysosomal activity on other inhibitors Using the method of the present invention, the concentration dependence of intracellular lysosomal activity on BafA1 and chloroquine (CQ) was confirmed in the same manner as in Example 2. HEK293 cells were cultured in a 6-well plate at 3 × 10 ５ The cells were diluted with DMEM medium to give 100 cells / well and seeded. The seeded cells were incubated at 37°C, 5% CO ２After 24 hours of incubation under these conditions, the culture supernatant was removed, and DMEM medium containing 100 nM BafA1 or 0.5, 5, or 50 μM CQ and 10 μg / mL DQ Green BSA was added and incubated at 37°C for 6 hours. As a control, a medium containing no inhibitor was added and incubated in the same manner. Subsequently, extract preparation, fluorescence measurement, background measurement, and protein concentration measurement were performed as in Example 1. The background was subtracted from the fluorescence measurement value of each sample and the result was divided by the protein concentration to calculate RFU. The RFU value of the control was set to 100%, and the relative lysosomal activity (%) was calculated from the RFU values ​​of each sample containing BafA1 or CQ and graphed (Figure 4). N = 3 samples were used for each group.

[0055] The results are shown in Figure 4. The error bars in the figure indicate standard error. It was shown that the measured lysosomal activity decreased in a concentration-dependent manner not only in the presence of BafA1 but also in the presence of CQ. Both BafA1 and CQ are drugs that have been reported to cause damage to lysosomes, and it was shown that the measured lysosomal activity obtained by the method of the present invention actually reflects lysosomal activity.

[0056] [Example 4] Measurement of lysosomal activity using various cell lysates Using HEK293 cells, lysosomal activity was measured using various cell lysates. HEK293 cells were plated in a 6-well plate at 3 × 10 ５ The cells were diluted with DMEM medium to give 100 cells / well and seeded. The seeded cells were incubated at 37°C, 5% CO ２ After culturing for 24 hours under the above conditions, the culture supernatant was removed, and DMEM medium containing 0 (control), 100 nM BafA1, and 10 μg / mL DQ Green BSA was added, followed by incubation at 37°C, 5% CO ２The cells were incubated at RT for 6 hours. The supernatant was removed and the cells were collected. RIPA buffer (50 mM Tris-HCl, 0.1% SDS, 0.5% DOC, 1% NP-40, 150 mM NaCl (pH 8.0)), CHAPS buffer (50 mM Tris-HCl, 150 mM NaCl, 1% CHAPS (pH 7.6)), or Triton X-100 buffer (50 mM Tris-HCl, 150 mM NaCl, 50 mM EDTA, 1% Triton X-100 (pH 7.6)) was added, followed by ice cooling for 10 minutes. Subsequently, the extract was collected, and fluorescence, background, and protein concentration measurements were performed in the same manner as in Example 1. The background was subtracted from the fluorescence measurement value of each sample, and the result was divided by the protein concentration to calculate RFU. The RFU value of the sample without BafA1 was set to 100%, and the relative lysosomal activity (%) was calculated from the RFU value of each sample with BafA1 added and graphed (Figure 5). N = 3 samples were collected for each group.

[0057] Lysosomal activity measured using various cell lysates with and without BafA1 is shown in Figure 5. A decrease in lysosomal activity due to BafA1 was confirmed regardless of the type of buffer used.

[0058] [Example 5] Measurement of intracellular lysosomal activity using DQ ovalbumin and cell lysate Using HEK293 cells, lysosomal activity was measured using DQ ovalbumin and cell lysate. HEK293 cells were plated in a 6-well plate at 3 × 10 ５ The cells were diluted with DMEM medium to give 100 cells / well and seeded. The seeded cells were incubated at 37°C, 5% CO ２ After culturing for 24 hours under these conditions, the culture supernatant was removed, and DMEM medium containing 0, 1, 10, and 100 nM BafA1 and 10 μg / mL DQ ovalbumin was added, followed by incubation at 37°C and 5% CO ２The cells were incubated at RT for 6 hours. Cell lysis, recovery of extract, fluorescence measurement, background measurement, and protein concentration measurement were then performed in the same manner as in Example 1. The background was subtracted from the fluorescence measurement value of each sample, and the result was divided by the protein concentration to calculate the RFU. The RFU value of the sample containing no BafA1 was set to 100%, and the relative lysosomal activity (%) was calculated from the RFU value of each sample to which BafA1 had been added, and the results were graphed ( FIG. 6 ). N=3 samples were collected for each group.

[0059] Lysosomal activity measured using DQ ovalbumin at various BafA1 concentrations is shown in Figure 6. Error bars in the figure indicate standard error. As with the use of DQ Green BSA, it was confirmed that lysosomal activity decreased in a BafA1 concentration-dependent manner when DQ ovalbumin was used.

[0060] The present invention can be used in the fields of medicine, pharmaceutical manufacturing, etc. All publications, patents, and patent applications cited in this specification are hereby incorporated by reference in their entirety.

Claims

1. A method for measuring intracellular lysosomal activity in vitro, comprising the following steps (1) to (4): (1) culturing the cells in a medium containing a fluorescent substance-binding protein; (2) recovering the cultured cells after step (1); (3) adding a cell lysis composition to the cultured cells recovered in step (2) to obtain a cell lysate fraction; and (4) measuring the fluorescence intensity of the cell lysate fraction obtained in step (3).

2. The method according to claim 1, further comprising steps (5) and (6) following step (4): (5) measuring the protein concentration of the cell lysate fraction; and (6) determining the value (RFU (Relative Fluorescence Units)) obtained by dividing the fluorescence intensity of step (4) by the protein concentration measured in step (5).

3. The method of claim 1, wherein the fluorescent substance is at least one selected from the group consisting of FITC, Cy3, Cy5 and BODIPY.

4. The method of claim 1, wherein said fluorescent substance is BODIPY and said protein is bovine serum albumin (BSA).

5. The method according to claim 1, wherein the cell lysis composition is at least one selected from the group consisting of CHAPS lysis buffer, HBST, cell lysis buffer, Buffer A, M-PER buffer, cell lysis buffer M, and RIPA buffer.

6. The method of claim 1, wherein the cells are at least one of HeLa cells, HEK293 cells, HEI-OC1 cells, and HUVECs.

7. A method for assisting in the assessment of the effectiveness of a disease treatment in a subject, comprising measuring intracellular lysosomal activity in a sample removed from the subject by the method of claim 1.

8. A kit for measuring intracellular lysosomal activity in vitro, comprising: a first measurement reagent solution containing a fluorescent substance-binding protein; and a second measurement reagent solution containing a composition for cell lysis.

9. The kit according to claim 8, further comprising a third measurement reagent for quantifying protein concentration.

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