CN120160961B - Novel probe combination for detecting cell metabolism through full spectrum flow and application thereof - Google Patents

Abstract

The invention relates to the field of medical detection, in particular to a probe combination for detecting cell metabolism through full spectrum flow and application thereof. Traditional flow cytometry is limited by overlapping fluorescent channels, and cannot detect multiple metabolic states in the same sample at the same time, so that detection efficiency and data accuracy are affected. According to the invention, through optimizing the probe combination and utilizing the full spectrum flow cytometry, synchronous detection of two or more metabolic probes can be realized, and simultaneously a plurality of metabolic indexes such as intracellular fatty acid intake, glucose intake, active oxygen level, mitochondrial function and the like are detected, and compatibility of fluorescent signals among the probes is ensured. The method can reduce sample consumption, improve detection efficiency, is suitable for metabolic analysis of trace clinical samples and rare cell populations, is widely applied to the fields of tumor research, metabolic disease monitoring, drug screening and the like, and provides an efficient and accurate technical means for single-cell level metabolic research.

Description

Novel probe combination for detecting cell metabolism through full spectrum flow and application thereof

Technical Field

The invention relates to the field of medical detection, in particular to a novel probe combination for detecting cell metabolism through full spectrum flow and application thereof.

Background

Metabonomics (Metabonomics/Metabolomics) is an emerging discipline developed at the end of the 90 s of the 20 th century, and is a science that studies the type, quantity and rules of variation of its metabolites (endogenous metabolites) after an organism has been disturbed (e.g. by genetic changes or environmental changes). Metabolism is the key to understanding various physiological and pathological processes, metabonomics research has become a research hotspot in the field of life science in recent years, and most of research objects are small molecular substances with relative molecular mass of less than 1000, including sugar metabolism, lipid metabolism, energy metabolism, amino acid metabolism, nucleic acid metabolism and the like. Currently, metabolic disorders have been shown to be closely related to many diseases such as diabetes, tumor, inflammation, etc., and it is important to adapt to environmental changes by adjusting the metabolic state of cells to exert their effect, and related papers are published in various authoritative academic journals such as Nature, medicine, journal ofHepatology, CANCER RESEARCH, etc.

Methods for single cell level metabolic detection are limited. The flow cytometry is used as a high-flux and high-sensitivity cell analysis technology, can be used for detecting metabolism related probes to evaluate mitochondrial functions and cell metabolism activities, becomes a sharp instrument for single-cell level metabolism research, and is more and more widely applied to metabolism research. Researchers can quickly and accurately acquire metabolic state information of cells and deeply explore the relationship between cell metabolism and occurrence and development of diseases. Common metabolism related probes include lipid metabolism fluorescent probes, glucose uptake fluorescent probes, mitochondrial related probes, active oxygen detection probes, lactate uptake probes, and the like. The technology relates to 11 kinds of fluorescent probes in total, and summarizes the fluorescent probes, corresponding fluorescent channels and detection objects in table 1, and the probes are specifically as follows:

1. sugar and lipid metabolism probes:

BODIPY C12 (4, 4-difluoro-5-methyl-4-boron-3 a,4 a-diaza-s-indacen-3-lauric acid) and BODIPY C16 (4, 4-difluoro-5, 7-dimethyl-4-boron-3 a,4 a-diaza-s-indacen-3-palmitic acid) are both BODIPY (4, 4-difluoro-boradiazaindacene, fluoroboro-dipyrrole compound) fluorescent labeled saturated fatty acids, λex/λem is about 488/515nm, which can penetrate cell membranes into the cell interior and carry out localization and specific staining on intracellular polar lipids. The fluorescent probe is often used for researches such as fatty acid uptake, lipid transport, absorption and accumulation of lipid in tissues such as fat or liver, and the like, is also used as a universal membrane probe for dyeing cells, and the like, can be used as a synthetic precursor of various fluorescent phospholipids, and is suitable for various fluorescent detection systems such as a fluorescent microscope, a laser confocal microscope, a flow cytometer, and the like.

NBD-cholesterol (NBD for short) is a fluorescent analogue of cholesterol containing fluorescent Nitrobenzoxadiazole (NBD) groups, λex/λem. Apprxeq.465/535 nm. The probe can be used for in vitro and in vivo steroid uptake and esterification analysis, intracellular cholesterol transport mechanism research, metabolism of mammals, bacteria and cells, and hamster intestinal tract cholesterol absorption research.

2-NBDG (2- (N- (7-nitrobenzo-2-oxa-1, 3-diazole-4-amino) -2-deoxyglucose) is a fluorescent-labeled deoxyglucose analog, λex/λem is about 465/540nm. The probe can accumulate in living cells but does not enter a glycolytic pathway, the fluorescence intensity generated by the probe is proportional to glucose uptake, and the glucose uptake of cells can be quantitatively detected, so that the probe is suitable for evaluating the glucose metabolic activity of various types of cells such as adipocytes, immune cells and cancer cells.

2. Mitochondrial function probes:

MitoTracker Green is a cell-penetrating carboanthocyanin derivative, which is a bright green fluorescent probe, λex/λem≡490/516nm. It fluoresces little in aqueous solution, only when it accumulates in the lipid environment of mitochondria, and its ability to localize to mitochondria is not affected by mitochondrial membrane potential, so it can be used for semi-quantitative detection of mitochondrial mass.

MitoTracker Deep Red is a far infrared fluorescent probe, λex/λem≡644/665nm, which is specifically used to label and detect mitochondria. It has high cell permeability and mitochondrial specificity, and can selectively mark the mitochondria with biological activity in living cells and detect mitochondrial membrane potential. Is suitable for multiple fluorescent staining experiments and long-time living cell imaging and dynamic observation. In addition, mitoTracker Deep Red can still keep good fluorescent marking effect after the cells are subjected to fixation by aldehyde fixative and permeabilization by detergent, so that the fluorescent marking agent has wide application value in the fields of cell biology research and the like.

Tetramethyl rhodamine ethyl ester (Tetramethylrhodamine ETHYL ESTER, TMRE) is a non-cytotoxic cell-permeable cationic fluorescent probe, and λex/λem is approximately equal to 549/575nm, and can specifically identify mitochondrial membrane potential. A large amount of negative charges exist in mitochondria in a normal state, TMRE can be gathered in a mitochondrial matrix after entering cells, and bright orange fluorescence is emitted; when the mitochondrial membrane potential decreases, TMRE is released into the cytoplasm and the intensity of the orange fluorescence in mitochondria decreases significantly. Therefore, TMRE can rapidly and sensitively detect changes in cell, tissue or purified mitochondrial membrane potential and can be used for early apoptosis detection.

3. Oxidative stress and active oxygen detection:

Reactive oxygen species (Reactive Oxygen Species, ROS) refers to a class of oxygen-containing compounds that are produced metabolically by the body and are highly reactive. ROS are normal byproducts of body metabolism, and the balance of ROS maintains normal physiological functions of cells. H2-DCFDA (dichlorofluorescein diacetate) is a chemically reduced fluorescein, λex/λem. Apprxeq.495/529 nm, commonly used as an indicator of ROS in cells. After the acetate group is cut and oxidized by cytolactonase, non-fluorescing H2-DCFDA is converted into 2',7' -Dichlorofluorescein (DCF) which emits high-intensity fluorescence, and the content and the change of active oxygen in cells can be judged by detecting the fluorescence of the DCF, so that the method is also suitable for various fluorescence detection systems such as a fluorescence microscope, a laser confocal microscope, a flow cytometer and the like.

Mitochondria are one of the main sources of ROS, and MitoSOX Red (mitochondrial superoxide indicator) is commonly used to detect mitochondrial ROS, the principle of which is the ability to specifically target mitochondria of living cells. The probe can be oxidized by superoxide after entering mitochondria, but not oxidized by other active oxygen and active nitrogen, and the oxidation product can emit strong fluorescence, and λex/λem is approximately 510/580nm, so that the superoxide in the mitochondria can be selectively detected.

DHE (dihydroethidine) is a fluorescent probe of superoxide anion which dehydrogenates to form ethidium bromide under the influence of intracellular superoxide anions. Ethidium bromide can be combined with RNA or DNA to generate red fluorescence, λex/λem is approximately 518/605nm, and the content of intracellular peroxide is detected by detecting the intensity of the red fluorescence, which acts similarly to H2-DCFDA.

4. Microenvironment sensing probe:

pHrodo Red (intracellular pH indicator) is a novel fluorescent probe for detecting the pH value in living cells. pHrodo Red exhibits weak fluorescence at neutral pH, but fluorescence gradually increases as pH decreases, λex/λem+.560/585 nm, an ideal tool for studying phagocytosis and its regulation by drugs and/or environmental factors. Extracellular fluorescence is absent, without the need for washing steps and quenching dyes.

Currently, staining based on traditional flow cytometry metabolic-related probes still has the following problems:

1. spectral overlap interference limited multi-parameter synchronous detection:

(1) The problem of mutual exclusion of homologous channel probes is that the fluorescence channels of a plurality of metabolic probes have obvious overlapping, such as 6 metabolic probes of BODIPY C12, BODIPY C16, mitoTracker Green, H2-DCFDA, NBD and 2-NBDG can be detected by using FITC channels in the traditional flow, and 4 metabolic probes of pHrodo Red, DHE, mitosox Red and TMRE can be detected by using PE channels in the traditional flow. This makes it impossible to find synergistic or antagonistic effects of different metabolic processes (such as dynamic association of sugar metabolism and lipid metabolism) in the same cell. In addition, the fractional detection introduces time variations that make it difficult to capture transient changes in metabolic state (e.g., temporal relationships of ROS burst and mitochondrial membrane potential collapse).

(2) The compatibility of the traditional fluorescein and the metabolic probe is conflicted, namely, the metabolic probe staining can be used for simultaneously marking cells with the traditional fluorescein, namely, the traditional fluorescein is used for marking different cell subgroups, and the metabolic probe is used for analyzing metabolic change characteristics in the different subgroups. If inexperienced, the metabolic probes are not properly matched with the traditional luciferin, and the situation that the metabolic related probes of the same channel cannot be used together with the traditional luciferin, such as BODIPY C12, BODIPY C16, mitoTracker Green, H2-DCFDA, NBD, 2-NBDG, FITC, AF488, and the like, and pHrodo Red, DHE, mitosox Red, TMRE, PE, and the like, can not be easily analyzed for the change of a single metabolic probe in different cell subsets at the same time.

2. Experimental efficiency and data reliability challenges:

(1) Sample consumption increases and experimental complexity increases, the single index detection mode forces the sample to increase exponentially (n times the number of probes), and reagent consumption (about 300-500 mu L/sample) and labor cost (2-3 times longer processing time) are significantly increased. Particularly, for micro clinical samples and rare cell populations, how to dye a plurality of probes with fewer samples to obtain more metabolic information is a need to be solved. In addition, the metabolic activity of living cells is susceptible to external environmental fluctuations, and delayed batch detection may introduce systematic errors, so that the acquired data cannot accurately reflect the initial metabolic characteristics of the sample.

(2) The clinical transformation value is limited, the occurrence, development and metabolism changes of a plurality of diseases in clinic are closely related, and the physiological and pathological changes of organism organs often cause the changes of in-vivo metabolism, so the detection of metabolites is very important for knowing metabolic diseases in time. However, the single-dimensional detection of the traditional method is difficult to reveal a panoramic network of a pathological mechanism, and the development of an individualized treatment strategy is restricted. The more convenient and rapid metabolic detection method is still further researched by researchers,

There is no relevant probe combination available at present.

Disclosure of Invention

Aiming at the problems in the prior art, a spectrum flow detection method is adopted to analyze whether probes with high overlapping degree of fluorescent channels and probes and traditional fluorescein are compatible, and through multiple experiments, 12 groups of co-channel double-probe combinations, 11 groups of co-channel fluorescein and probe combinations and 2 groups of multi-generation Xie Tanzhen combinations are obtained, so that two or more metabolic probes are simultaneously used for single-tube sample staining. The sample and economic cost are greatly saved, so that the simultaneous analysis of the metabolism functions of single cell sugar intake, lipid intake, active oxygen level, mitochondrial membrane potential and the like of a trace clinical sample and rare cell population becomes possible.

Specifically, the invention provides the following technical scheme:

In a first aspect, the invention provides a compatible full spectrum flow assay for the detection of cellular metabolism, the probe combination being selected from the group consisting of BODIPY C12 and MitoTracker Green, BODIPY C12 and H2-DCFDA, BODIPY C12 and 2-NBDG, BODIPY C16 and H2-DCFDA, BODIPY C16 and NBD, BODIPY C16 and 2-NBDG, mitoTracker Green and H2-DCFDA, mitoTracker Green and 2-NBDG, H2-DCFDA and 2-NBDG, pHrodo Red and Mitosox Red, DHE and TMRE, mitosox Red and TMRE.

In one embodiment, BODIPY C12 and MitoTracker Green, BODIPY C12 and H2-DCFDA, BODIPY C12 and 2-NBDG, BODIPY C16 and H2-DCFDA, BODIPY C16 and NBD, BODIPY C16 and 2-NBDG, mitoTracker Green and H2-DCFDA, mitoTracker Green and 2-NBDG, H2-DCFDA and 2-NBDG are used simultaneously as FITC channels, and pHrodo Red and Mitosox Red, DHE and TMRE, mitosox Red and TMRE are used simultaneously as PE channels.

In one embodiment, the probe combination is compatible for use with fluorescein FITC, AF488, PE, APC or AF 647.

In a preferred embodiment, AF488 is compatible with BODIPY C16, NBD or 2-NBDG, FITC is compatible with BODIPY C16, NBD or 2-NBDG, PE and pHrodo Red, DHE, mitosox Red or TMRE, and APC and MitoTracker Deep Red are compatible.

In a second aspect, the invention provides a cell metabolism detection method based on the probe combination, wherein after a cell sample is stained by using the probe combination, metabolic parameters in the cell sample are analyzed simultaneously by adopting full spectrum flow cytometry.

In one embodiment, the method is to simultaneously detect multiple metabolic activities including, but not limited to, lipid metabolism, glucose metabolism, reactive oxygen species, mitochondrial function, and cell membrane potential within the same cell sample.

In one embodiment, the cells are labeled with a fluorescent antibody and a combination of probes is used with an antibody fluorescein (e.g., FITC, AF488, PE, APC, AF 647) to effect metabolic profiling of a particular cell subset.

In a preferred embodiment, the probe combination is H2-DCFDA, mitosox Red and TMRE.

In a preferred embodiment, the probe combination is BODIPY C12, 2-NBDG, mitosox Red, TMRE.

In a more preferred embodiment, the probe combination is used in combination with a fluorescently labeled antibody.

Drawings

The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention. In the drawings:

FIG. 1 is a spectrum of 11 probes;

FIG. 2 is a flow chart showing the separation of the two probes of BODIPY C12, BODIPY C16, mitoTracker Green, H2-DCFDA, NBD and 2-NBDG, and FIG. 2B is a chart showing the similarity and separation results between the 6 probes;

FIG. 3 is a diagram showing the separation flow of the probes of pHrodo Red, DHE, mitosox Red and TMRE, and FIG. 3B is a diagram showing the similarity and separation results between the 4 probes;

FIG. 4 is a diagram showing the analysis of combinations of compatible luciferins (FITC, AF488, PE, APC and AF 647) and co-channel probes, wherein FIG. 4A shows the flow chart of the separation of the luciferins FITC, AF488 from the co-channel probes BODIPY C12, BODIPY C16, mitoTracker Green, H2-DCFDA, NBD and 2-NBDG, FIG. 4B shows the similarity and separation results between the above-mentioned luciferins and probes, FIG. 4C shows the flow chart of the separation of PE from the co-channel probes pHrodo Red, DHE, mitosox Red and TMRE, FIG. 4D shows the similarity and separation results between the above-mentioned luciferins and probes, FIG. 4E shows the flow chart of the separation of the APCs AF647 from the co-channel probes MitoTracker Deep Red, and FIG. 4F shows the similarity and separation results between the above-mentioned luciferins and probes;

FIG. 5 is a graph of H2-DCFDA, mitoSOXRed and TMRE flow splitting results;

FIG. 6 is a graph showing the correlation of the average fluorescence intensities of samples of the rotenone/antimycin A stimulated group and the control group, and the 3 kinds of probe co-stained groups;

FIG. 7 is a graph of H2-DCFDA, mitoSOXRed, TMRE and MitoTracker Deep Red flow split results;

FIG. 8 is a graph showing the correlation of the average fluorescence intensities of samples of the rotenone/antimycin A stimulated group and the control group, and the 4 kinds of probe co-stained groups;

FIG. 9 is a graph of BODIPYC, 2-NBDG, mitoSOXRed, and TMRE flow splitting results;

FIG. 10 is a high-dimensional analysis of human peripheral blood mononuclear cells after staining for a total of 19 subpopulations;

FIG. 11 is a graph showing the correlation of the average fluorescence intensity of the single probe staining and 4 probe co-staining groups of different subpopulations of 19 lymphocytes.

Detailed Description

The preferred embodiments of the present invention will be described below with reference to the accompanying drawings, it being understood that the preferred embodiments described herein are for illustration and explanation of the present invention only, and are not intended to limit the present invention.

Example 1 screening of Probe combinations

1. And (3) establishing a probe spectrum, namely respectively carrying out 11 kinds of probe staining on the A2780 single-cell suspension, carrying out on-line acquisition signals by using a full spectrum flow cytometer (SONY ID 7000) to obtain spectrum information of the 11 kinds of probes, and introducing the spectrum information into a spectrum library (figure 1).

2. FITC channel-under compatible probe combinations were explored and shown in Table 1, wherein the FITC channel-under probes included BODIPYC, BODIPYC, mitoTracker Green, H2-DCFDA, NBD, 2-NBDG, and were 6 in total.

Table 111 probe fluorescent channel and test article

First, the similarity between probes can be initially queried from FluoroFinder web sites, as shown in fig. 2B. The experimental verification is carried out after that, the cultured A2780 cells are made into single cell suspension and counted, 7x 10 ⁶ cells are resuspended in 700 mu LPBS and are split into 7 EP tubes, one of the tubes is not dyed, the other 6 tubes are respectively added with BODIPY C12, BODIPY C16, mitoTracker Green, H2-DCFDA, NBD and 2-NBDG according to the proportion, after incubation for 30min at 37 ℃ in the absence of light, 1mL PBS is added to stop dyeing, 300g centrifugation is carried out for 5min, 200 mu L PBS is added to resuspend and filter. The 6 stained samples were mixed pairwise in equal volume and an equal volume of unstained sample was added, and the on-machine detection was performed using a full spectrum flow cytometer (SONY ID 7000) and the flow diagram as shown in fig. 2A was obtained. The horizontal and vertical axes represent two groups of cell positive signals "horizontal and vertical", meaning that the two fluorescent probes can be split and used simultaneously, e.g., BODIPY C12 and 2-NBDG, while the antisense cannot be used simultaneously, e.g., BODIPY C12 and BODIPY C16.

Thus we have obtained 9 compatible probes and 6 incompatible probes under the FITC channel. The 9 compatible sets of probes were BODIPY C12 and MitoTracker Green, BODIPY C12 and H2-DCFDA, BODIPY C12 and 2-NBDG, BODIPY C16 and H2-DCFDA, BODIPY C16 and NBD, BODIPY C16 and 2-NBDG, mitoTracker Green and H2-DCFDA, mitoTracker Green and 2-NBDG, H2-DCFDA and 2-NBDG. The 6 incompatible probes were BODIPY C12 and BODIPY C16, BODIPY C12 and NBD, BODIPY C16 and MitoTracker Green, mitoTracker Green and NBD, H2-DCFDA and NBD, NBD and 2-NBDG.

3. The PE down-channel compatible probe combinations were explored, as shown in Table 1, and the PE down-channel probes included 4 types of pHrodo Red, DHE, mitosox Red, TMRE.

First, the similarity between probes can be initially queried from FluoroFinder web sites, as shown in fig. 3B. The experimental verification is carried out after that, the cultured A2780 cells are made into single cell suspension and counted, 5x10 ⁶ cells are resuspended in 500 mu LPBS and are split into 5 EP tubes, one of the tubes is not dyed, the other 4 tubes are respectively added with pHrodo Red, DHE, mitosox Red and TMRE according to a proportion, incubated at 37 ℃ for 30min in a dark place, 1mL PBS is added to stop dyeing, 300g centrifugation is carried out for 5min, 200 mu L PBS is added to resuspend and filtration is carried out. The 4 stained samples were mixed pairwise in equal volume and the equal volume of unstained sample was added, and the on-machine detection was performed using a full spectrum flow cytometer (SONY ID 7000) and the flow chart shown in fig. 3A was obtained. The horizontal and vertical axes represent two groups of cell positive signals "horizontal and vertical", meaning that the two fluorescent probes can be split and used simultaneously, e.g., mitoSOXRed and TMRE, and the antisense cannot be used simultaneously, e.g., mitosox Red and pHrodo Red.

From this we have obtained 3 sets of compatible probes and 3 sets of incompatible probes under the PE channel, as shown in FIG. 3. The 3 compatible sets of probes are pHrodo Red and MitoSOXRed, DHE and TMRE, mitoSOXRed and TMRE, and the 3 incompatible sets of probes are pHrodo Red and DHE, pHrodo Red and TMRE, DHE and MitoSOXRed.

4. Exploring compatibility of probes with co-channel luciferins (FITC, AF488, PE, APC and AF 647)

First, the similarity between probes and corresponding channel luciferins can be initially queried from FluoroFinder website, as shown in fig. 4-B, D, F. After that, experiments were carried out to verify that 5 1.5mL EP tubes were prepared, 1 drop (50. Mu.L) of UltraComp eBeads ^TM Plus compensation microspheres were added dropwise to each tube, fluorescent antibodies of FITC, AF488, PE, APC and AF647 were added respectively, incubated for 15min in the dark, centrifuged for 5min at 300g, and resuspended in 200. Mu.L PBS. A2780 cell (staining procedure with 2) of single-stained BODIPY C12, BODIPYC, mitoTracker Green, H2-DCFDA, NBD, 2-NBDG was mixed with FITC, AF 488-stained compensation microspheres, A2780 cell (staining procedure with 3) of single-stained pHrodo Red, DHE, mitoSOXRed, TMRE was mixed with PE-stained compensation microspheres, A2780 cell of single-stained MitoTracker Deep Red was mixed with APC, AF 647-stained compensation microspheres, and on-machine detection was performed using a full spectrum flow cytometer (SONYID 7000) and a flow chart as shown in FIG. 4A, C, E was obtained.

As can be seen, the following luciferins are compatible with the probe combinations AF488 and BODIPY C16, AF488 and NBD, AF488 and 2-NBDG, FITC and BODIPY C16, FITC and NBD, FITC and 2-NBDG, PE and pHrodo Red, PE and DHE, PE and MitoSOXRed, PE and TMRE, APC and MitoTracker Deep Red. Fluorescein was not compatible with the probe combinations AF488 and BODIPY C12, AF488 and MitoTracker Green, AF488 and H2-DCFDA, FITC and BODIPYC12, FITC and MitoTracker Green, FITC and H2-DCFDA, AF647 and MitoTracker Deep Red.

Further, we selected the most commonly used two combinations from the compatible probe combinations for verification, depending on the experimental use. It should be noted that the combination of the probes of the present invention is not limited to the probe combinations of example 2 and example 3, and those skilled in the art can select and use the compatible probe combinations from the compatible probe combinations according to different experimental purposes.

Example 2 verification of the detection Effect of the H2-DCFDA, mitoSOXRed, TMRE Probe combination

This example is used to demonstrate simultaneous staining of multiple metabolic probes (H2-DCFDA, mitoSOXRed, TMRE) in a single tube and evaluation of intracellular reactive oxygen species, mitochondrial membrane potential levels of tumor cell A2780 after simultaneous stimulation with rotenone and antimycin A. By comprehensively analyzing the three indexes, the functions of intracellular active oxygen and mitochondria can be comprehensively and accurately estimated, and richer information is provided for researching the physiological and pathological processes of cells.

The experimental method comprises the following steps:

1. and (3) verifying compatibility among probes:

Example 1 (3) has demonstrated that probes Mitosox Red and TMRE under the PE channel are detachable, we will further demonstrate that probe H2-DCFDA under the FITC channel is detachable from Mitosox Red, TMRE. The method comprises preparing tumor cell A2780 sample to be tested into single cell suspension, counting, taking 4×10 ⁶ cells, re-suspending in 400 μL PBS, sub-packaging in 4 EP tubes, adding H2-DCFDA, mitosox Red and TMRE into the rest 3 tubes respectively according to a certain proportion, incubating at 37deg.C for 30min in dark, adding 1mL PBS, stopping staining, centrifuging for 5min at 300g, adding 200 μL PBS, and re-suspending and filtering. The 3 stained samples and the unstained sample were mixed in equal volumes and were tested on-line using a full spectrum flow cytometer (SONY ID 7000).

2. And (3) verifying the detection effect of the probe combination:

3 kinds of probes are simultaneously stained, namely, a sample after stimulation of rotenone and antimycin A and a sample of a control group are respectively prepared into single cell suspensions and counted, 1x10 ⁶ cells or required cell amounts are respectively taken and resuspended in 100 mu L of PBS, and 4 tubes of cells are prepared for each sample. H2-DCFDA, mitosox Red, TMRE and 3 probes are respectively added according to the proportion for co-staining, after incubation for 30min at 37 ℃ in the dark, 1mL PBS is added for stopping staining, 300g centrifugation (different centrifugal force sizes are selected according to different cells) is carried out for 5min, and 200 mu L PBS is added for resuspension and filtration.

The on-board detection was performed using a full spectrum flow cytometer (SONY ID 7000).

Results:

From FIG. 5 we can see that 3 metabolic probe combinations based on full spectrum flow cytometry H2-DCFDA, mitosox Red and TMRE can be split and used for staining the same tube samples. When we carried out correlation analysis on the average fluorescence intensities of single-staining and co-staining samples of the rotenone/antimycin A stimulation group and the control group, the results show (figure 6) that the correlation between the 3-probe co-staining group and the single-probe staining group is remarkable, and the consistency is strong, which means that one tube of sample can simultaneously carry out analysis and evaluation on the total active oxygen level, the mitochondrial active oxygen level and 3 metabolic functions of mitochondrial membrane potential of cells.

Example 3 detection Effect verification of BODIPY C12, 2-NBDG, mitosox Red, TMRE Probe combination

This example illustrates the use of a combination of metabolic probes (BODIPY C12, 2-NBDG, mitosox Red, TMRE) in combination with fluorescent-labeled antibodies in a single tube to evaluate sugar uptake, lipid uptake, mitochondrial reactive oxygen species, mitochondrial membrane potential levels in individual lymphocyte subsets of Peripheral Blood Mononuclear Cells (PBMC).

The experimental method comprises the following steps:

1. and (3) verifying compatibility among probes:

Example 1 (2) has demonstrated that the probes BODIPY C12, 2-NBDG under the FITC channel are resolvable, example 1 (3) has demonstrated that the probes Mitosox Red and TMRE under the PE channel are resolvable, and we will further demonstrate that the 4 probes described above are resolvable. The method comprises preparing sample to be tested into single cell suspension, counting, taking 4×10 ⁶ cells, re-suspending in 400 μ LPBS, sub-packaging in 5 EP tubes, adding BODIPY C12, 2-NBDG, mitosox Red and TMRE into the rest 3 tubes respectively, incubating at 37deg.C for 30min in dark, adding 1mL PBS, stopping staining, centrifuging for 5min at 300g, adding 200 μl PBS, and re-suspending and filtering. The 4 stained samples and the unstained samples were mixed in equal volumes and were tested on-line using a full spectrum flow cytometer (SONY ID 7000).

2. And (3) verifying the detection effect of the probe combination:

surface staining and probe simultaneous staining:

First, 14 surface markers of lymphocytes were selected, compatible fluorescein and probe combinations were selected according to example 1, and FITC and AF488 fluorescein incompatible with BODIPY C12 were not selected, and specific staining indexes are shown in table 2.

Table 2 pbmc 14 surface markers and corresponding luciferins

The specific staining method is to separate and count human peripheral blood mononuclear cells (PERIPHERAL BLOOD MONONUCLEAR CELL, PBMC) by adopting a density gradient centrifugation method, and to take 1x10 ⁷ cells for 14 surface markers staining of lymphocytes. The cells were then resuspended in 500. Mu.L PBS and aliquoted into 5 EP tubes for BODIPY C12, 2-NBDG, mitosox Red, TMRE staining and 4 probes co-staining, respectively.

The on-machine detection was performed using a full spectrum flow cytometer (SONY ID 7000), and the data analysis was performed using FlowJo software.

Results:

From FIG. 9 we can see that the 4 metabolic probe combinations based on full spectral flow cytometer BODIPYC, 2-NBDG, mitosox Red and TMRE are compatible and can be used for staining the same tube samples. When we stained PBMCs with 14 surface markers in combination with 4 probes, the results were subjected to both decreasing-dimension and clustering analysis to obtain 19 different subpopulations (fig. 10), and the uniformity of the average fluorescence intensity of the clustered 19 different subpopulations single probe-stained and 4 probe co-stained groups was compared, and the correlation between the two groups was analyzed. The results showed (FIG. 11) that the correlation between the 4 probe co-stained groups and the single probe stained groups was significant and consistent.

Comparative examples H2-DCFDA, mitoSOXRed, TMRE and MitoTracker Deep Red detection Effect of the combination of probes

We explored the H2-DCFDA, mitosox Red, TMRE and MitoTracker Deep Red probe combinations in the same way, and found that the 4 probes were methodically compatible (FIG. 7), but when the average fluorescence intensity correlation analysis (FIG. 8) was performed for single and co-stained samples of rotenone/antimycin A stimulated and control groups, an abnormality occurred in MitoTracker Deep Red, demonstrating that these 4 probes could not be used simultaneously.

It will be apparent to those skilled in the art that modifications and variations can be made in the combination of the invention according to the needs of the experiment without departing from the spirit and scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

Claims (9)

1. A full spectrum flow detection method for cell metabolism is characterized by comprising the steps of simultaneously analyzing metabolic parameters in a cell sample by adopting full spectrum flow cytometry after staining the cell sample by using compatible probe combinations, wherein the probe combinations are selected from the group consisting of BODIPY C12 and MitoTracker Green, BODIPY C12 and H2-DCFDA, BODIPY C12 and 2-NBDG, BODIPY C16 and H2-DCFDA, BODIPY C16 and NBD, BODIPY C16 and 2-NBDG, mitoTracker Green and H2-DCFDA, mitoTracker Green and 2-NBDG, H2-DCFDA and 2-NBDG, pHrodo Red and Mitosox Red, DHE and TMRE, mitosox Red and TMRE;

wherein BODIPY C12 and MitoTracker Green, BODIPY C12 and H2-DCFDA, BODIPY C12 and 2-NBDG, BODIPY C16 and H2-DCFDA, BODIPY C16 and NBD, BODIPY C16 and 2-NBDG, mitoTracker Green and H2-DCFDA, mitoTracker Green and 2-NBDG or H2-DCFDA and 2-NBDG are used simultaneously under the FITC channel, and pHrodo Red and Mitosox Red, DHE and TMRE or Mitosox Red and TMRE are used simultaneously under the PE channel.

2. The method of claim 1, wherein the probe combination is compatible with fluorescein FITC, AF488, PE, APC, or AF 647.

3. The method of claim 2, wherein AF488 is compatible with BODIPY C16, NBD or 2-NBDG, FITC is compatible with BODIPY C16, NBD or 2-NBDG, PE is compatible with pHrodo Red, DHE, mitosox Red or TMRE, and APC is compatible with MitoTracker Deep Red.

4. The method of claim 3, wherein the method is for simultaneously detecting multiple metabolic activities including lipid metabolism, glucose metabolism, reactive oxygen species, mitochondrial function and cell membrane potential within the same cell sample.

5. The method of claim 3 or 4, wherein the probe combination is used simultaneously with fluorescein and in combination with fluorescent antibodies to cell surface markers to achieve metabolic profiling of specific cell subsets.

6. The method of claim 5, wherein the fluorescein is FITC, AF488, PE, APC, or AF647.

7. The method of claim 3 or 4, wherein the probe combination is H2-DCFDA, mitoSOX Red, and TMRE.

8. The method of claim 3 or 4, wherein the probe combination is BODIPY C12, 2-NBDG, mitoSOX Red, and TMRE.

9. The method of claim 8, wherein the probe combination is used in combination with a fluorescently labeled antibody.