Method for determining lysosomal activity

Abstract

Provided is a method with which it is possible to easily measure lysosomal activity of cells in a sample. A method for measuring the activity of lysosome in a cell in vitro, said method comprising the following steps (1)-(4): (1) a step for culturing a cell by adding a fluorescent substance-binding protein to a cell-containing medium; (2) a step for collecting the cultured cells after step (1); (3) a step for adding a composition for cell lysis to the cultured cells collected in step (2) to obtain a cell lysate component; and (4) a step for measuring the fluorescence intensity of the fluorescent substance-bound protein in the cell lysate component obtained in step (3).

Description

Technical Field

[0001] This invention relates to a method for determining lysosomal activity. Background Technology

[0002] Lysosomes are small intracellular organelles. Their membrane-bound interiors are acidic, and they are characterized by their enzymatic activity in breaking down various biological macromolecules through the action of proteases, glycosidases, lipases, nucleases, and other enzymes. Because lysosomes are the endpoints of multiple pathways such as endocytosis and autophagy, they are known to play a wide range of physiological roles, directly or indirectly.

[0003] Autophagy is one of the protein breakdown pathways associated with lysosomes. Autophagy is a mass breakdown system ubiquitous in eukaryotic cells, functioning to maintain cellular homeostasis by constantly replacing cytoplasmic components. When autophagy is induced in response to various stimuli, vesicles surrounded by double membranes (i.e., autophagosomes) fuse with lysosomes to form autophagic lysosomes, within which unwanted proteins are broken down. It is known that abnormal protein breakdown function of these autophagic lysosomes can lead to pathological states in various diseases such as cancer, neurodegenerative diseases, cardiovascular diseases, lung diseases, and infectious diseases, and may also trigger vital phenomena such as aging and impaired motor function. Autophagy has been reported to play a particularly important role in the maintenance of hearing and cell morphology.

[0004] Various methods for measuring lysosomal activity within cells have existed since ancient times, but a unified method has yet to be established (Patent Document 1). Classic methods for detecting protease activity in lysosomes include cathepsin maturity assays using pulse tracking with radioactive isotopes and immunoblotting using anti-cathepsin antibodies. While these methods are widely used, the former requires the use of radioactive isotopes, and the latter is prone to errors due to variations in cell density, probe concentration, and uptake rate. Furthermore, a method using a lysozyme-tracking dye that targets the acidic contents of lysosomes is known, but it has not been used for quantitative determination (Non-Patent Document 1). Methods for measuring lysosomal activity have been reported, such as quantitative assays using fluorescent probes embedded in plasmids and plasmid-based lysosomal activity assays using flow cytometry, but these are not readily applicable (Patent Document 1, Non-Patent Document 2). These methods are complex to operate, and if performed by an unskilled operator, different results may be obtained with each measurement, making it difficult to obtain reliable results.

[0005] As a method for measuring lysosomal activity, the following method (Non-Patent Literature 3) has been developed. This method involves the intracellular degradation of DQ (trademark)-BSA, a fluorescent dye formed by binding BODIPY to bovine serum albumin, and the emitted fluorescence is measured under a microscope. Because this method requires microscopic observation, obtaining high-precision and reliable results necessitates further research into image manipulation, acquiring reliable fluorescence intensity data, background removal, and improving reusability, in addition to sample processing. Therefore, it is not a simple measurement method.

[0006] Furthermore, all the aforementioned existing methods are based on measurements performed in cultured cells. While this type of analysis using cultured cells is a compelling and powerful tool for selective visualization, such as examining the intracellular localization of target substances, microscopic observation requires specialized equipment and is complex to operate. Moreover, because it is limited to analysis within a finite field of view, it is necessary to verify whether it accurately reflects the whole picture. In addition, the viability of the target cells, batch-to-batch reproducibility, and the influence of the presence or absence of culture medium must also be considered. Existing technical documents Patent documents

[0007] Patent Document 1: International Publication No. 2011 / 019082 Non-patent literature

[0008] Non-patent literature 1: Cytometry Part A, 85A; 169-178, 2014 Non-patent literature 2: Sci. Rep. 9; 11635, 2019 Non-patent literature 3: Bio Protoc. 7(19); e2571, 2017 Summary of the Invention The technical problem that the invention aims to solve

[0009] The purpose of this invention is to provide a simple method for determining the lysosomal activity of cells in a sample without the need for microscopic observation of cells. Technical means to solve technical problems

[0010] In view of the above, the inventors conducted repeated and in-depth research and found that by adding fluorescent substances to bind proteins, recovering cultured cells from the cell culture container, dissolving the cells with cell lysate and measuring the fluorescence intensity, a stable and high-precision lysosomal activity assay can be easily performed without being affected by interfering substances and without relying on cell observation.

[0011] That is, the present invention provides the following content. [1] A method for in vitro determination of intracellular lysosomal activity, the method comprising the following steps (1) to (4): (1) The process of culturing the cells in a culture medium containing a fluorescent substance-binding protein; (2) The process of recovering the cultured cells after process (1); (3) The step of adding a cell lysing composition to the cultured cells recovered in step (2) to obtain a cell lysate component; and (4) A process for measuring the fluorescence intensity of the cell lysate components obtained in process (3). [2] The method as described in [1], wherein the method further includes steps (5) and (6) after step (4): (5) Procedure for determining the protein concentration of cell lysate components; (6) The step of 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 as described in [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 of any one of [1] to [3], wherein the fluorescent substance is BODIPY and the protein is bovine serum albumin (BSA). [5] The method of any one of [1] to [4], wherein the cell lysis composition is selected from at least one of 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 any one of [1] to [5], wherein the cell is at least one of HeLa cells, HEK293 cells, HEI-OC1 cells and HUVEC. [7] A method for assisting in evaluating the effectiveness of treatment for a subject’s disease, the method comprising determining intracellular lysosomal activity in a sample extracted from the subject by means of the method of claim 1. [8] A kit for in vitro determination of intracellular lysosomal activity, the kit comprising: The first assay solution containing a fluorescent substance binding to a protein; and The second assay solution contains a cell lysis composition. [9] The kit as described in [8], wherein the kit further comprises a third assay solution for quantifying protein concentration. This specification contains the disclosure of Japanese Patent Application No. 2023-182873, which forms the basis of the priority claim of this application. Beneficial effects

[0012] According to the present invention, a method for easily determining the lysosomal activity of cells in a sample without the need for microscopic observation of cells can be provided. Attached Figure Description

[0013] Figure 1 This is a graph showing, as a comparative example, the results of measuring lysosomal activity in HEK293 cells in the presence of BafA1 by co-staining with DQ Green BSA and DAPI. The experiment was performed three times on different dates, with N=4 each time. The error bars in the graph represent the standard error. Figure 2 This is a graph illustrating the results of measuring lysosomal activity in HEK293 cells in the presence of BafA1, as in Example 1, by staining with DQ Green BSA and lysing the cells with cell lysate. The experiment was performed three times on different dates, with N=3 each time. The error bars in the graph represent the standard error. Figure 3 This is a graph illustrating the results of measuring lysosomal activity in HeLa cells, HEI-OC1 cells, and HUVECs at various concentrations of BafA1, as in Example 2, by staining with DQ Green BSA and lysing cells with cell lysis buffer. A represents lysosomal activity in HeLa cells, B represents lysosomal activity in HE1-OC1 cells, and C represents lysosomal activity in HUVECs. The experiment was performed with N=3. The error bars in the graph represent the standard error. Figure 4 This is a graph illustrating the results of measuring lysosomal activity in HEK293 cells, as in Example 3, by staining with DQ Green BSA and lysing the cells with cell lysate in the presence of BafA1 or various concentrations of CQ. Each assay was performed with N=3. The error bars in the graph represent the standard error. Figure 5 This is a graph showing the results of measuring lysosomal activity in HEK293 cells using CHAPS buffer and Triton X-100 buffer as Example 4. The experiment was performed with N=3. The error bars in the graph represent the standard error. Figure 6 This is a graph illustrating the results of measuring lysosomal activity in HEK293 cells in the presence of BafA1, as in Example 5, by staining with DQ ovalbumin and lysing the cells with cell lysate. The experiment was performed with N=3. The error bars in the graph represent the standard error. Detailed Implementation

[0014] 1. Structure and Definition <Sample> In this specification, "sample" is not particularly limited as long as it includes cells that can be used as the object of lysosomal activity assay, including all organs, tissues, and cells. Examples include organs, tissues, and cells from vertebrates such as mammals, birds, reptiles, amphibians, and fish. Mammals are preferred, and humans or laboratory animals (mice, rats, hamsters, rabbits, etc.) are more preferred. "Cells" as used herein are not particularly limited as long as they contain proteins, and can be in any form, such as cell lines, primary cultured cells, and tissue sections, and can come from any specimen. In particular, cultured cells can be used appropriately. Furthermore, "tissue" as used herein is not particularly limited, and can include, for example, cancerous tissue, ischemic tissue, or tissue cultured under special conditions. Samples can be collected from tissues or cells, or from the environment of tissues or cells. In some examples, samples can be tissue biopsy material, blood, plasma, extracellular fluid, cultured cells, culture medium, waste tissue, plant material, synthetic proteins, archaea, bacteria, hyphae, or protozoa. Examples of cultured cells and primary cultured cells include 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), but are not limited to these.

[0015] <Fluorescent substance-binding protein> In this specification, "fluorescent substance-binding protein" refers to a protein bound to a fluorescent substance (also known as a "self-quenching dye"), and specifically to a protein that emits fluorescence through partial hydrolysis after being taken up by lysosomes. There are no particular limitations on the proteins constituting "fluorescent substance-binding proteins," but bovine serum albumin (BSA) is preferred. In this specification, "fluorescent substance" refers to organic compounds, proteins, or other substances that emit fluorescence of a specific wavelength when irradiated with light of a specific excitation wavelength. As organic compounds, examples include FITC (manufactured by Molecular Probes), Texas Red (manufactured by Molecular Probes), Cy3 and Cy5 (manufactured by GE HealthCare), 4,4-difluoro-4-borona-3a,4a-diaza-s-indarin (BODIPY), boron dipyrrole methylene dyes such as BDP-FL (BODIPY dyes), BIP, CF-MONO, CF-BI, BDPFL NHS-Ester, etc. Furthermore, as proteins, examples include GFP, CFP, RFP, and YFP. Preferably, BODIPY FL and BODIPY TR can be used.

[0016] As a "protein bound to a fluorescent substance," commercially available products can also be used, for example. Examples of such commercially available products include DQ-BSA (manufactured by Thermo Fisher Scientific), which is formed by binding BODIPY to BSA.

[0017] <Composition for Cell Dissolution> In this specification, the term "composition for cell lysis" refers to any solution composition commonly used in cell lysis or protein solubility. Examples include known substances such as CHAPS lysis buffer, RIPA buffer, HBST, cell lysis buffer, tissue lysis buffer, and buffer A. These are also commonly referred to as "cell lysis reagents" or "cell lysis solutions." These can be appropriately selected and used by those skilled in the art. The composition for cell lysis may contain a buffer. As a buffer, substances with buffering properties near weak bases, such as PBS, Tris, and HEPES, can be used.

[0018] To make proteins soluble, cell lysis compositions may contain surfactants such as CHAPS, SDS, NP40, and Triton-X. Additionally, urea, which also has soluble properties, may be included, and its use can be selected appropriately.

[0019] To prevent protein degradation caused by proteases in cells, protease inhibitors can be included. Commonly used protease inhibitors can be selected from PMSF (phenylmethylsulfonyl fluoride), aprotinin, leucopeptide, pepsin inhibitors, sodium fluoride, sodium orthovanadate, etc. Furthermore, divalent metal ions can be used as blocking agents (chelating agents) to inhibit proteases. Examples of chelating agents include EDTA or EGTA. Additionally, to maintain the phosphorylation state of proteins, phosphatase inhibitors such as sodium fluoride, sodium orthovanadate, sodium pyrophosphate, and β-glycerophosphate can be used.

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

[0021] The additives contained in the cell lysis composition can be appropriately selected and prepared for use by those skilled in the art. For example, in the case of RIPA buffer, it 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 these compositions and prepare for use according to the experimental purpose. In addition, for example, commercially available M-PER buffer (manufactured by Thermo Fisher Scientific) and cell lysis buffer M (manufactured by Fujifilm and Wako Pure Chemicals Co., Ltd.) can also be used. Preferably, RIPA buffer can be used.

[0022] In this specification, "kit" refers to an assembly of multiple elements used to carry out a method. In this specification, "reagent" refers to a substance consisting of a single element. Here, a test solution contained in a container constitutes one element.

[0023] 2. Methods for determining intracellular lysosomal activity The 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, characterized by comprising the following steps (1) to (4). (1) The process of culturing the cells in a culture medium containing a fluorescent substance-binding protein; (2) The process of recovering the cultured cells after process (1); (3) The step of adding a cell lysing composition to the cultured cells recovered in step (2) to obtain a cell lysate component; and (4) A step of measuring the fluorescence intensity of the fluorescent substance-bound protein in the cell lysate component obtained in step (3).

[0024] The method of this embodiment, when measuring lysosomal activity within cells, includes a cell-dissolving step, enabling quantitative measurement of lysosomal enzyme activity without complex operations such as image analysis. Therefore, lysosomal activity can be measured simply and accurately regardless of the user's skill level.

[0025] <Process (1) Cultivation Process> The method of this embodiment will be described below step by step. The following will exemplarily describe the steps for measuring lysosomal activity using HEK293 cells, but this is not intended to limit the scope of the invention.

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

[0027] The pre-cultured medium is replaced with a medium containing fluorescently bound proteins, and cell culture continues. Here, the concentration of the fluorescently bound proteins is not particularly limited and can be 1 μg / mL to 100 μg / mL, 5 μg / mL to 50 μg / mL, or especially around 10 μg / mL. The preferred culture temperature is 30℃ to 40℃, particularly 32℃ to 38℃. The preferred culture time is 10 minutes to 24 hours, particularly 30 minutes to 8 hours. The culture temperature and time can be adjusted appropriately according to the type and properties of the cells used.

[0028] <Process (2) Recycling Process> Step (2) of the method in this embodiment is a step of recovering the cultured cells after step (1). When recovering the cells, a protease such as trypsin can be used depending on the type of cells. For example, the cultured cells can be recovered by centrifuging at 4°C, 1000×g (approximately 2000 rpm) for 5 minutes. Here, the cells can be washed with ice-cooled PBS and the number of cells can be counted.

[0029] <Process (3) Cell Dissolution Process> Step (3) of the method in this embodiment is a step of adding a cell lysis composition to the cultured cells recovered in step (2) to obtain a cell lysate component. Here, as a cell lysis composition, a step using RIPA buffer is described by way of example.

[0030] Add the cooled RIPA buffer to the cells recovered in step (2). The RIPA buffer preferably contains a protease inhibitor. For 10^6 cells recovered, 100 μL of RIPA buffer is preferred, but the amount can be adjusted depending on the cell type, conditions, or the expected higher protein concentration. The amount of RIPA buffer used can be appropriately selected by those skilled in the art. The RIPA buffer containing the cells can then be repeatedly mixed by vortexing and stored on ice for 30 minutes before being used for lysosomal activity assays.

[0031] When the cells used for the assay are derived from tissue, the tissue can be dissected. If blood removal is necessary, it can be done by washing with ice-cooled PBS. While cooling on ice, the tissue is cut into small pieces and placed in a homogenizer with RIPA buffer containing protease inhibitors. The amount of RIPA buffer used at this stage is, for example, 500 μL for approximately 10 mg of tissue. The RIPA buffer containing tissue is completely homogenized using the homogenizer, and after standing on ice for 30 minutes, it is used for lysosomal activity assays. If necessary, a sonication process can be performed. In the sonication process, the sample is sonicated to further disrupt cells or tissue and cleave DNA. The sonication time can be adjusted appropriately depending on the type of sample. For example, while cooling the sample on ice, a reference standard can be set at 180 watts, with cell lysis for 1 minute and tissue lysis for 2-5 minutes (repeated in cycles of 10 seconds of sonication / 10 seconds of stillness).

[0032] <Process (4) Fluorescence Measurement Process> Step (4) of the method of this embodiment is a step of measuring the fluorescence intensity of the fluorescent substance-binding protein of the cell lysate component obtained in step (3). There are no particular limitations on the method for measuring the fluorescence intensity of the cell lysate component; existing methods such as ELISA readers and flow cytometry can be used appropriately. For ease of and short-time measurement, it is preferable to use an ELISA reader to measure the fluorescence intensity. Hereinafter, a step using an ELISA reader is described by way of example.

[0033] The cell lysate component extracted using the cell lysate composition is added to a black 96-well plate for luminescent fluorescence analysis. The fluorescence intensity is measured using a microplate reader (e.g., BMG Labtech) (excitation wavelength 485 nm, fluorescence wavelength 520 nm when using DQ-BSA), thereby determining lysosomal activity. The measurement can be performed once per sample or multiple measurements can be used. Multiple measurements are preferred for more accurate lysosomal activity determination. Lysosomal activity can be more accurately determined by correcting the measured fluorescence intensity using factors such as protein concentration. For example, the fluorescence intensity of the RIPA buffer used as background can be subtracted from the fluorescence intensity.

[0034] <Process (5) Protein Concentration Determination Process> The method of this embodiment may further include steps (5) and (6) after step (4). Step (5) of the method of this embodiment is the step of determining the protein concentration of the cell lysate components. There are no particular limitations on the method for determining the protein concentration; any known method may be used, for example, it may be carried out by the general method of using the BCA method. The BCA method can be used to quantify the protein using the Pierce BCA Protein Assay Kit (manufactured by ThermoFisher Scientific).

[0035] <Process (6) RFU Calculation Process> Step (6) of the method in this embodiment is to calculate the value (RFU, relative fluorescence unit) obtained by dividing the fluorescence intensity of step (4) by the protein concentration measured in step (5). By expressing lysosomal activity in RFU, for example, when evaluating the effect of drugs acting on lysosomes, it is possible to more accurately assess whether there is a change in lysosomal activity.

[0036] <Application Example> The method of this embodiment, by having the above-described structure and process, can easily measure intracellular lysosomal activity, and therefore is expected to contribute to research on lysosomal storage disorders, various diseases considered related to autophagy such as cancer and neurodegenerative diseases, aging, and motor function. Furthermore, the method of this embodiment can be effectively used in the following situations: evaluating the drug concentration dependence of diseases, screening therapeutic drugs, and as an indicator to confirm the rationality of treatment strategies for various diseases. In particular, it can be used as a method for monitoring the effectiveness of treatment in evaluating the effectiveness of disease management.

[0037] As an example of the application of the method of this embodiment, a method for screening drugs with lysosomal dysfunction recovery capabilities is given. The inventors have discovered that by coexisting cells with a lysosomal dysfunction agent, intracellular lysosomal activity measured by the method of this embodiment decreases in a manner dependent on the concentration of the lysosomal dysfunction agent. This indicates that the method of this embodiment can quantitatively evaluate the degree of lysosomal dysfunction and the degree of its recovery. This screening method may, for example, include the following steps (a) to (d). (a) The process of culturing cells using a culture medium containing candidate drugs, lysosomal disruptors and proteins bound to fluorescent substances; (b) The process of recovering cultured cells after process (a); (c) The step of adding a cell lysing composition to the cultured cells recovered in step (b) to obtain a cell lysate component; and (d) A step of measuring the fluorescence intensity of the fluorescent substance-bound protein in the cell lysate component obtained in step (c). By confirming the extent to which the lysosomal activity reduced in step (a) due to the action of the lysosomal inhibitor is restored by the candidate drug through steps (b) to (d), the ability of the candidate drug to restore lysosomal activity can be easily confirmed.

[0038] As "lysosomal disruptors," bafilomycin A1 (hereinafter sometimes referred to as "BafA1") and chloroquine (hereinafter sometimes referred to as "CQ"), which are known lysosomal disruptors, can be used. In addition, any substance with lysosomal disrupting activity can be used, such as hydroxychloroquine, which has a structure similar to chloroquine, azithromycin and other dibenzylisoquinoline alkaloids, and tetrandrine and other macrolide antibiotics.

[0039] 3. Auxiliary methods for evaluating the effectiveness of disease treatment. The second embodiment of the present invention is a method for assisting in the evaluation of the effectiveness of treatment for a subject's disease. The method of this embodiment is characterized by including: measuring intracellular lysosomal activity in a sample extracted from the subject using the method of the first embodiment. In the method of this embodiment, the reagents, equipment, samples, and conditions used are the same as in the first embodiment unless otherwise specified and unless there are significant contradictions.

[0040] Intracellular lysosomal activity can serve as an indicator of the rationality of treatment strategies for various diseases and is used to evaluate the effectiveness of treatments. It is known that lysosomes contain a large number of enzymes and related proteins; functional abnormalities such as decreased activity of these enzymes can lead to lysosomal diseases such as sphingolipidosis, mucopolysaccharidosis, oligosaccharidosis, glycogenopathies, neutral lipid storage diseases, and I-cell diseases (mucopolysaccharidosis II). These are more than 40 congenital metabolic disorders that cause various symptoms and are considered "designated difficult diseases" in Japan. The method of this embodiment can be used to obtain an indicator of the rationality of treatment strategies for diseases, referring to lysosomal diseases and the like, which are known to be highly correlated with cellular lysosomal activity. For example, lysosomal activity can be measured in vitro on samples extracted from the subject, and compared with control values, thereby serving as an indicator for determining the increase or decrease of the dosage of therapeutic drugs.

[0041] For example, a prescribed dose of therapeutic drug can be administered to a patient suffering from a disease known for its high correlation with lysosomal activity. The patient's intracellular lysosomal activity can be measured and compared to a pre-set threshold or a control value before treatment administration, allowing for adjustments to the dosage. For instance, if lysosomal activity is low compared to a pre-set threshold or control value, a higher dosage can be administered. Conversely, if lysosomal activity is high compared to a pre-set threshold or control value, a lower dosage can be administered.

[0042] 4. Reagent kit for lysosomal activity assay The third embodiment of the present invention is a kit for in vitro determination of intracellular lysosomal activity. The kit of this embodiment is characterized by comprising: a first assay solution containing a fluorescent substance-binding protein; and a second assay solution containing a cell lysis composition. More specifically, the kit of this embodiment is a kit for use with the method of the first embodiment. In the kit of this embodiment, the reagents, equipment, samples, and operating conditions used are the same as in the first embodiment unless otherwise specified and unless there is a significant contradiction.

[0043] The kit of this embodiment comprises at least two elements: a first assay solution containing a fluorescent substance-binding protein and a second assay solution containing a cell lysis composition. The first assay solution may be a culture medium containing a fluorescent substance-binding protein. Alternatively, the first assay solution may be a buffer solution containing a high concentration of a fluorescent substance-binding protein. The second assay solution may be a cell lysis composition (e.g., RIPA buffer), or a 5× solution of the cell lysis composition (for dilution before use), etc.

[0044] The kit of this embodiment can also include a composition for quantifying protein concentration as a third assay solution. For quantifying protein concentration, any known composition for protein concentration quantification can be used. For example, in the case of quantifying protein by the BCA method, a diquinoline carboxylic acid solution and a copper sulfate solution can be included.

[0045] In addition, the kit of this embodiment may also include, as needed, cell culture dishes, black microplates for fluorescence assay, and instruction manuals (attached documents). Example

[0046] [Comparative Example] Determination of intracellular lysosomal activity using co-staining with DQ Green BSA and DAPI HEK293 cells were cultured in DMEM medium at a concentration of 2 × 10⁻⁶. 4 Cells were diluted per well and seeded onto 8-well slides. After incubating at 37°C and 5% CO2 for 24 hours, the culture supernatant was removed, and DMEM medium containing 0 nM (control), 200 nM Bafilomycin A1 (BafA1, Bioaustralis), and 10 μg / mL DQ Green BSA was added. The cells were incubated at 37°C for 6 hours. The supernatant was removed, cells were fixed with 1% paraformaldehyde, and stained with DAPI. Images were captured using a fluorescence microscope (KEYENCE) (exposure time: GFP 4 seconds, DAPI 1 / 200 second). The threshold was standardized to 19-255 using ImageJ, and the area of ​​green fluorescence was calculated. A graph was plotted using the value calculated by dividing this value by the number of DAPI-stained cell nuclei. Figure 1 Each group was sampled at N=4. Three trials were conducted on different implementation dates. The error bars in the figure represent the standard error.

[0047] The results of the three tests are shown in Figure 1The measurements varied significantly across different implementation dates, making it impossible to definitively detect the decrease in lysosomal activity caused by BafA1 addition. This is believed to be due to the fact that, during imaging using fluorescence microscopy, the field of view within the wells was manually selected, leading to significant inter-operator and inter-sample variability, resulting in insufficient accuracy and reproducibility.

[0048] [Example 1] Determination of intracellular lysosomal activity using DQ Green BSA and cell lysate HEK293 cells were cultured in DMEM medium at a concentration of 3 × 10⁻⁶. 5 Cells were diluted per well and seeded into 6-well plates. After incubating the seeded cells at 37°C and 5% CO2 for 24 hours, the culture supernatant was removed, and DMEM medium containing 0 nM (control), 100 nM BafA1, and 10 μg / mL DQ Green BSA was added. The cells were incubated at 37°C for 6 hours. The supernatant was removed, the cells were recovered, and RIPA buffer (50 mM Tris-HCl, 0.1% SDS, 0.5% DOC, 1% NP-40, 150 mM NaCl (pH 8.0)) was added. The cells were then chilled on ice for 10 minutes. Intracellular proteins were extracted to obtain the extract.

[0049] The extract was dispensed at 150 μL / well into black 96-well plates for luminescent fluorescence analysis, and the fluorescence intensity was measured using a microplate reader (BMG Labtech, Inc.) (excitation wavelength 485 nm, fluorescence wavelength 520 nm). Furthermore, the proteins contained in the extract were quantified using the Pierce BCA protein assay kit (Thermo Fisher Scientific, Inc.).

[0050] The fluorescence intensity of the RIPA buffer used as background was subtracted from the fluorescence intensity of each BafA1 concentration sample, and then divided by the protein concentration (RFU) in the extract. Samples were taken in groups of N=3. Three experiments were performed on different implementation dates.

[0051] The results of the three tests are shown in Figure 2 The error bars in the figure represent the standard error. This shows that the errors for each implementation day are small, and highly reproducible results are obtained compared to Comparative Example 1. Furthermore, it shows that the decrease in lysosomal activity caused by BafA1 can be reliably observed. This is believed to be because, unlike measurements using fluorescence microscopy, batch measurements of the signal can be performed without the need for manual field-of-view selection.

[0052] [Example 2] Dependence of lysosomal activity assay on BafA1 concentration The dependence of lysosomal activity assays on BafA1 concentration was confirmed using HeLa cells, HEI-OC1 cells, and HUVECs. HeLa cells, HEI-OC1 cells, and HUVECs were cultured in DMEM at a concentration of 3 × 10⁶ cells / mL. 5 Cells were diluted per well and seeded into 6-well plates. After 24 hours of culture, HeLa cells and HUVECs were cultured at 37°C and 5% CO2, while HEI-OC1 cells were cultured at 33°C and 10% CO2. The culture supernatant was then removed. HeLa cells and HEI-OC1 cells were supplemented with DMEM medium containing 0 nM, 1 nM, 10 nM, and 100 nM BafA1 and 10 μg / mL DQ Green BSA. HUVECs were supplemented with DMEM medium containing 0 nM, 0.1 nM, 1 nM, and 10 nM BafA1 and 10 μg / mL DQ Green BSA. HeLa cells and HUVECs were incubated at 37°C for 6 hours, and HEI-OC1 cells were incubated at 33°C for 1 hour. Subsequently, the preparation of the extract, fluorescence measurement, background measurement, and protein concentration determination were performed in the same manner as in Example 1. The RFU (Recovery Frequency) was calculated by subtracting the background value from the fluorescence measurement value of each sample and dividing by the protein concentration. The RFU value of samples without BafA1 was set to 100%. Based on the RFU values ​​of samples with BafA1, the relative lysosomal activity (%) was calculated and plotted. Figure 3 Each group was sampled with N=3.

[0053] The dependence of lysosomal activity in different cells on BafA1 concentration is shown in... Figure 3 . Figure 3 A shows the results for HeLa cells. Figure 3 B shows the results for HEI-OC1 cells. Figure 3 Figure C shows the results for HUVEC. The error bars in the figure represent the standard error. As shown, it was confirmed that lysosomal activity decreased in a BafA1 concentration-dependent manner in HeLa cells, HEI-OC1 cells, and HUVECs. This demonstrates that the method of the present invention can detect barriers to lysosomal activity dependent on drug concentration, as well as differences in cellular drug sensitivity.

[0054] [Example 3] Concentration-dependent inhibitors of lysosomal activity Using the method of this invention, following the same steps as in Example 2, the concentration-dependent relationship between intracellular lysosomal activity and BafA1 and chloroquine (CQ) was confirmed. HEK293 cells were cultured in DMEM medium at a concentration of 3 × 10⁻⁶. 5Cells were diluted per well and seeded into 6-well plates. After incubating the seeded cells at 37°C and 5% CO2 for 24 hours, the culture supernatant was removed, and DMEM medium containing 100 nM BafA1 or 0.5 μM, 5 μM, or 50 μM CQ and 10 μg / mL DQ Green BSA was added. The cells were incubated at 37°C for 6 hours. As a control, cells in culture with no inhibitors were incubated in the same manner. Subsequently, the preparation of the extract, fluorescence measurement, background measurement, and protein concentration determination were performed in the same manner as in Example 1. The RFU (Reactive Fluorescence Usage) was calculated by subtracting the background value from the fluorescence value of each sample and dividing by the protein concentration. The RFU value of the control was set to 100%. Based on the RFU values ​​of each sample containing BafA1 or CQ, the relative lysosomal activity (%) was calculated and plotted. Figure 4 Each group was sampled with N=3.

[0055] The results are shown in Figure 4 The error bars in the figure represent the standard error. This shows that lysosomal activity assays decrease 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 reported to cause lysosomal dysfunction, demonstrating that the lysosomal activity assays obtained by the method of this invention substantially reflect lysosomal activity.

[0056] [Example 4] Lysosomal activity assay using various cell lysates Lysosomal activity was measured using HEK293 cells with various cell lysing solutions. HEK293 cells were cultured in DMEM at a concentration of 3 × 10⁶ cells / mL. 5Cells were diluted per well and seeded into 6-well plates. After incubating the seeded cells at 37°C and 5% CO2 for 24 hours, the culture supernatant was removed, and DMEM medium containing 0 nM (control), 100 nM BafA1, and 10 μg / mL DQ GreenBSA was added. The cells were incubated at 37°C and 5% CO2 for 6 hours. The supernatant was removed, and the cells were recovered. 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)), and Triton X-100 buffer (50 mM Tris-HCl, 150 mM NaCl, 50 mM EDTA, 1% Triton X-100 buffer (pH 7.6)) were added, and the cells were cooled on ice for 10 minutes. Subsequently, the extraction, fluorescence, background, and protein concentration were determined in the same manner as in Example 1. The RFU (Reactive Fluorescence Function) was calculated by subtracting the background value from the fluorescence value of each sample and dividing by the protein concentration. The RFU value of samples without BafA1 was set as 100%. Based on the RFU values ​​of samples with BafA1, the relative lysosomal activity (%) was calculated and plotted. Figure 5 Each group was sampled with N=3.

[0057] Lysosomal activity, measured using various cell lysates with and without BafA1, is shown in... Figure 5 The results showed that regardless of the type of buffer used, the decrease in lysosomal activity was confirmed to be caused by BafA1.

[0058] [Example 5] Determination of intracellular lysosomal activity using DQ ovalbumin and cell lysate Lysosomal activity was measured using HEK293 cells with DQ ovalbumin and cell lysate. HEK293 cells were cultured in DMEM at a concentration of 3 × 10⁶ cells / year. 5 Cells were diluted per well and seeded into 6-well plates. After incubating the seeded cells at 37°C and 5% CO2 for 24 hours, the culture supernatant was removed, and DMEM medium containing 0 nM, 1 nM, 10 nM, and 100 nM BafA1 and 10 μg / mL DQ ovalbumin was added. The cells were then incubated at 37°C and 5% CO2 for 6 hours. Subsequently, cell lysis, extraction recovery, fluorescence measurement, background measurement, and protein concentration determination were performed in the same manner as in Example 1. The RFU (Relative Lysosomal Activity) was calculated by subtracting the background from the fluorescence measurement value of each sample and dividing by the protein concentration. The RFU value of samples without BafA1 was set as 100%. Based on the RFU values ​​of samples with added BafA1, the relative lysosomal activity (%) was calculated and plotted. Figure 6 Each group was sampled with N=3.

[0059] Lysosomal activities at various BafA1 concentrations, as determined using DQ ovalbumin, are shown below. Figure 6 The error bars in the figure represent standard errors. It was confirmed that, similar to the case with DQ Green BSA, lysosomal activity also decreased in a BafA1 concentration-dependent manner when using DQ ovalbumin. Industrial applicability

[0060] This invention can be used in the medical field, pharmaceutical manufacturing field, etc. All publications, patents and patent applications cited in this specification are incorporated herein by reference.

Claims

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

2. The method as described in claim 1, wherein, The method further includes steps (5) and (6) after step (4): (5) Procedure for determining the protein concentration of cell lysate components; (6) The process of calculating the relative fluorescence unit (RFU) by dividing the fluorescence intensity of step (4) by the protein concentration measured in step (5).

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

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

5. The method of claim 1, wherein, The cell lysis composition is selected from at least one of 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 evaluating the effectiveness of treatment for a subject's disease, the method comprising determining intracellular lysosomal activity in a sample extracted from the subject by the method of claim 1.

8. A kit for in vitro determination of intracellular lysosomal activity, the kit comprising: The first assay solution containing a fluorescent substance binding to a protein; and The second assay solution contains a cell lysis composition.

9. The kit according to claim 8, wherein, The kit further includes a third assay solution for quantifying protein concentration.

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