CN116087069A - Method for detecting histone methylation and acetylation modification level of specific cell population based on flow cytometry - Google Patents
Method for detecting histone methylation and acetylation modification level of specific cell population based on flow cytometry Download PDFInfo
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Abstract
The invention discloses a method for detecting histone methylation and acetylation modification levels of specific cell populations based on flow cytometry, which uses formaldehyde and ethanol as fixatives, screens out fluorescent channels which are not affected by the fixatives, designs a proper combination of the fluorescent channels and antibodies, and detects the histone methylation and acetylation modification levels of cell samples to be detected through flow cytometry. The invention is based on flow cytometry, replaces the traditional detection method with low flux and high cost such as chromatin co-immunoprecipitation, western blot, immunofluorescence and the like, detects the methylation and acetylation modification level of the histone, can be specific to a specific cell group, and meets the requirements of scientific researchers on detecting the methylation and acetylation modification level of the histone on various cells such as T cells, macrophages or B cells and the like.
Description
Technical Field
The invention belongs to the technical field of biological medicine, and particularly relates to a method for detecting histone methylation and acetylation modification levels of specific cell populations based on flow cytometry.
Background
Histones (Histones), which mainly include H1, H2A, H2B, H, and H4, are a group of evolutionarily very conserved basic proteins found in the chromatin of eukaryotic cells, and have the effects of maintaining DNA structure, protecting genetic information, and regulating gene expression. Under the catalysis of methyltransferase, the N-terminal arginine and lysine residues of histones H3 and H4 are methylated. Histone methylation modification has the functions of regulating gene transcription, DNA repair, heterochromatin formation and the like, and the regulation result depends on methylation residues and methylation degree. Histone methylation modification abnormality is closely related to occurrence of diseases such as tumor.
In addition to methylation modification, histone lysine residues are acetylated under the action of Histone Acetyl Transferases (HATs) and can be deacetylated by deacetylase (HDACs). Histone acetylation modification has important significance: 1. neutralizing histone charge, weakening interaction of histone and DNA, loosening chromatin structure, facilitating transcription; 2. providing specific anchoring sites for transcription factors, facilitating binding; 3. the acetylation modification is combined with other modification modes to regulate gene transcription. Currently, histone deacetylase inhibitors (HDACi) are useful as mood stabilizers and antiepileptic drugs, and have been used as potential treatments for cancer, neurodegenerative diseases, and inflammatory diseases.
Therefore, by researching histone methylation and acetylation modification, not only can the apparent genetic control be more understood, but also a huge application prospect can be mined, and a mechanism research is provided for related drug development.
Currently, in the prior art, detection methods for histone methylation and acetylation modification mainly comprise chromatin co-immunoprecipitation, western blot, immunofluorescence and the like. However, each method has a certain degree of limitation, and cannot be balanced in sensitivity, detection cost, sample size, and accurate quantification. For example, chromatin co-immunoprecipitation is cumbersome, requires a long time span, requires the use of specialized equipment, and the difference in analytical methods can result in difficulty in balancing accuracy, simplicity, operational complexity, and cost; protein electrophoresis (Western blot) can only perform semi-quantitative detection on a low-flux sample on the whole level, and cannot meet the deeper requirements; immunofluorescence is not easy, and is not suitable for high-flux sample detection while being incapable of accurately quantifying. The comparison of the features of several common detection methods is shown in Table 1.
TABLE 1 characterization comparison of common histone methylation and acetylation modification level detection methods
Therefore, based on the limitations of the existing detection methods, a new detection method is needed in the art to meet the requirements of histone methylation and acetylation modification detection, that is, the method has the characteristics of high sensitivity, accurate quantification, simple operation, low detection cost, flexibility and the like.
Disclosure of Invention
The conventional flow staining procedure used the intracellular staining method using a commercial eBioscience ™ Foxp 3/transcription factor staining buffer kit. However, in the course of histone methylation and acetylation modification detection, the real level of histone methylation and acetylation modification cannot be detected in real time due to the presence of methyltransferases, acetyltransferases, and the like. Thus, there is a need to inactivate enzymes using formaldehyde and ethanol immobilization to detect the true level of histone methylation and acetylation modifications.
Thus, it is desirable to find suitable fluorescent channels that are not affected by fixatives, and to design suitable fluorescent channel and antibody combinations to determine flow cytometry detection methods for different immune cell populations.
In order to solve the technical problems, the invention provides a method for detecting histone methylation and acetylation modification levels of specific cell populations based on flow cytometry, which uses formaldehyde and ethanol as fixatives to detect the histone methylation and acetylation modification levels of cell samples to be detected by flow cytometry, and comprises the following steps:
step 1, preparing a fixing agent: preparing 1% formaldehyde as a pre-fixing agent and preparing 70% ethanol as a fixing agent;
step 2, screening a fluorescent channel to be detected: firstly, using BD cell fixing liquid as a fixing agent, detecting the fluorescence intensity and the expression percentage of a CD3 antibody under each fluorescent channel, then fixing a sample by using the fixing agent obtained in the step 1, detecting the fluorescence intensity and the expression percentage of the CD3 antibody under each fluorescent channel, comparing the results, and screening out fluorescent channels which are not influenced by the components of the fixing agent for subsequent detection;
and 4, performing flow staining on the sample according to the combination mode designed in the step 3, and measuring the histone methylation and acetylation modification level of the sample by adopting a flow cytometry.
Specifically, in step 2, the fluorescent channel to be measured includes BUV395, BUV496, BUV737, BV421, BV605, BV711, BV786, BV510, FITC, PE, APC and AF700.
Specifically, in step 3, the antibodies include CD45, CD3, CD4, CD8, CD11B, CD19, CD25, F4/80, NK1.1, foxp3 and actyl-Histone H2B (Lys 5) (D5H 1S) XP.
Specifically, in step 3, the cell population includes CD 4T cells, CD 8T cells, regulatory T cells (tregs), B cells, NK cells, and macrophages.
Specifically, in the combination mode of methylation detection, a first antibody and a second antibody are further arranged, wherein the first antibody is Di-Methyl-Histone H3 (Lys 9) (D85B 4) XP-rabit mAb, and the second antibody is Anti-rabit IgG (H+L) and F (ab') 2 Fragment.
Specifically, in the combination mode of methylation detection, under the condition of T cells, B cells and NK cell populations, the combination mode of fluorescent channels and antibodies is as follows:
BUV395 fluorescein coupled CD45 antibody, BUV496 fluorescein coupled CD4 antibody, BUV737 fluorescein coupled CD8 antibody, BV605 fluorescein coupled CD19 antibody, BV711 fluorescein coupled CD25 antibody, BV786 fluorescein coupled NK1.1 antibody, FITC fluorescein coupled CD3 antibody, PE fluorescein coupled Foxp3 antibody.
Specifically, in the combination mode of the acetylation detection, the combination mode of the fluorescent channel and the antibody under the T cell, B cell and NK cell population is as follows:
BUV395 fluorescein coupled CD45 antibody, BUV496 fluorescein coupled CD4 antibody, BUV737 fluorescein coupled CD8 antibody, BV605 fluorescein coupled CD19 antibody, BV711 fluorescein coupled CD25 antibody, BV786 fluorescein coupled NK1.1 antibody, FITC fluorescein coupled CD3 antibody, APC fluorescein coupled Foxp3 antibody, PE fluorescein coupled Acetyl-Histone H2B (Lys 5) (D5H 1S) XP antibody.
Specifically, in the combination mode of methylation detection, the combination mode of fluorescent channels and antibodies under the macrophage population is as follows:
BUV395 fluorescein coupled CD45 antibody, BV510 fluorescein coupled F4/80 antibody, BV711 fluorescein coupled CD19 antibody, FITC fluorescein coupled CD3 antibody, AF700 fluorescein coupled CD11b antibody.
Specifically, in the combination mode of the acetylation detection, the combination mode of the fluorescent channel and the antibody under the macrophage population is as follows:
BUV395 fluorescein coupled CD45 antibody, BV510 fluorescein coupled F4/80 antibody, BV711 fluorescein coupled CD19 antibody, FITC fluorescein coupled CD3 antibody, AF700 fluorescein coupled CD11B antibody, PE fluorescein coupled Acetyl-Histone H2B (Lys 5) (D5H 1S) XP Rabbit antibody.
Compared with the prior art, the invention has the beneficial effects that:
1) The method provided by the invention adopts formaldehyde pre-fixation and ethanol fixation methods, inhibits the activity of enzymes in a sample, and is convenient for detecting the real level of histone methylation and acetylation modification in real time.
2) The method provided by the invention effectively screens out fluorescent channels which are not influenced by fixatives such as ethanol and the like, designs a combination scheme of various antibodies, and further detects histone methylation and acetylation modification levels of different cell populations.
3) The method provided by the invention is used for carrying out qualitative and quantitative analysis on the sample from the cellular level based on flow cytometry, so that the problem that the methylation and acetylation modification levels of total histones in the sample can only be detected in the traditional technology is solved, the method can be applied to the methylation and acetylation modification levels of histones in various samples such as tumor tissues, spleen tissues and a specific cell population in blood samples, has the advantages of flexible and simple operation, higher sensitivity, lower detection cost and the like, and improves the detection flux, the quantitative detection level and the cost within a controllable range compared with the conventional methods such as chromatin immunoprecipitation, western blot, immunofluorescence and the like.
Drawings
FIG. 1 is a graph showing the results of the immune cell grouping in mouse spleen cells in example 1 of the present invention;
FIG. 2 is a graph showing the results of the detection of histone methylation modification levels on various immune cells in spleen cells of mice in example 1 of the present invention;
FIG. 3 is a graph showing the results of detecting the level of histone acetylation modification on various immune cells in spleen cells of a mouse according to example 1 of the present invention;
FIG. 4 is a graph showing the results of the methylation modification levels of B cell histones in spleen cells of mice before and after LPS stimulation in example 1 of the present invention;
FIG. 5 is a graph showing the results of the level of acetylation modification of macrophage tissue protein in spleen cells of mice before and after LPS stimulation in example 1 of the present invention.
Detailed Description
The following description of the present invention will be made clearly and fully, and it is apparent that the embodiments described are some, but not all, of the embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The experimental procedures, which do not address the specific conditions in the examples below, are generally carried out under conventional conditions or under conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated. The experimental materials involved in the invention are all commercial reagents unless specified, and are available from commercial sources. All that is referred to in the following examples, not specifically illustrated, are commercial reagents, wherein BUV395 CD3, BUV496 CD3, BUV737 CD3 are purchased from BD Biosciences; BV421 CD3, BV510 CD3, BV605 CD3, BV711 CD3, BV786 CD3, FITC CD3, perCP Cy5.5 CD3, PE CD3, PE-Cy7 CD3, APC CD3, AF700 CD3, APC-Cy7 CD3 were purchased from BioLegend. BD CytofixTM fixation buffer Anhui Ann's food Limited and Thermo fisher scientific were purchased from BD Biosciences, respectively, as absolute ethanol and ebiosciences (10X) permeabilized solution.
Example 1: detection of cell group protein methylation and acetylation modification levels in mouse spleen
According to the method for detecting histone methylation and acetylation modification levels of specific cell populations based on flow cytometry, formaldehyde and ethanol are used as fixatives, and the histone methylation and acetylation modification levels of a cell sample to be detected are detected by flow cytometry, which comprises the following steps:
step 1, preparing a fixing agent: preparing 1% formaldehyde as a pre-fixing agent and preparing 70% ethanol as a fixing agent;
step 2, screening a fluorescent channel to be detected: firstly, using BD cell fixing liquid as a fixing agent, detecting the fluorescence intensity and the expression percentage of a CD3 antibody under each fluorescent channel, then fixing a sample by using the fixing agent obtained in the step 1, detecting the fluorescence intensity and the expression percentage of the CD3 antibody under each fluorescent channel, comparing the results, and screening out fluorescent channels which are not influenced by the components of the fixing agent for subsequent detection; step 3, grouping sample cells according to the expression intensity of immune cell markers, and designing a combination mode of a fluorescent channel and an antibody under different cell populations, wherein the combination mode comprises a methylation detection combination mode and an acetylation detection combination mode;
and 4, performing flow staining on the sample according to the combination mode designed in the step 3, and measuring the histone methylation and acetylation modification level of the sample by adopting a flow cytometry.
In this example, when screening for fluorescent channels to be tested, the fluorescent channels and antibodies were combined as follows:
BUV395 CD3, BUV496 CD3, BUV737 CD3 purchased from BD Biosciences; BV421 CD3, BV510 CD3, BV605 CD3, BV711 CD3, BV786 CD3, FITC CD3, perCP Cy5.5 CD3, PE CD3, PE-Cy7 CD3, APC CD3, AF700 CD3, APC-Cy7 CD3.
In step 2, the test results using two fixing methods are shown in table 2 below:
TABLE 2 Effect of different fixatives on CD3 detection results at different fluorescent channels
From the results shown in Table 2, it can be seen that the channels PerCP-Cy5.5, PE-Cy7 and APC-Cy7 are greatly affected in the two fixing modes of 1% formaldehyde and 70% ethanol and BD fixing solution provided by the invention, which are unfavorable for the subsequent detection, so that the antibody combination design is not performed.
The sample cells were grouped according to the expression level of immune cell markers as follows:
CD4+ T cells (CD 45) + CD3 + CD4 + ) CD8+ T cells (CD 45) + CD3 + CD8 + ) Regulatory T (Treg) cells (CD 45 + CD3 + CD4 + CD25 + Foxp3 + ) B cells (CD 45) + CD3 - CD19 + ) NK cells (CD 45) + CD3 - NK1.1 + ) Macrophages (CD 45) + CD3 - CD19 - CD11b + F4/80 + ）。
In this example, lipopolysaccharides (LPS) were used to stimulate mouse spleen cells, i.e. to extract mouse spleen cells, and after 24 hours of stimulation with LPS at a final concentration of 900 ng/mL, flow staining was performed to compare the differences in methylation and acetylation modification levels of different cell populations, as follows:
a. mouse spleen cell extraction: the spleen was placed on a 70 μm filter screen, a spleen sample was piston-milled using a 2 mL syringe, and pre-chilled PBS was added to wash the sample while milling, and spleen cells were filtered into a 50 mL centrifuge tube, and centrifuged to obtain spleen cell pellets. Add 1 x red blood cell lysate, incubate at room temperature for 2 minutes to lyse red blood cells, terminate the reaction with pre-chilled PBS, and centrifuge resuspended. Spleen cells were counted and adjusted to the appropriate density.
b. LPS stimulates spleen cells: will be 1 x 10 6 Spleen cells were plated in 6-well plates, followed by stimulation of spleen cells with 900 ng/mL LPS and incubated in an incubator for 24 hours.
c. Histone methylation and acetylation staining: spleen cells of mice before and after LPS stimulation were collected and centrifuged, and cells were resuspended with PBS, counted and adjusted to a concentration of 5X 10 5 Individual cells/100 μl, plated into 96V-well plates at 100 μl/well, centrifuged at 4 ℃ for 5 min at 450 g, and the supernatant removed; cells were resuspended in 100 μl of PBS containing BV421 Live/read dye (0.1 μl per well), incubated at 4 ℃ for 30 min in the dark, centrifuged at 450 g for 5 min, and the supernatant removed; the cells were then resuspended with 100 μl staining buffer; mu.L of Mouse Fc Block (murine Fc blocker) was added to each well and incubated at 4℃for 5 min in the absence of light;
extracellular antibodies were added according to the antibody and fluorescent channel combinations designed in tables 3-6 below:
table 3: t cell, B cell and NK cell histone methylation antibodies and fluorescent combination protocol
Table 4: t cell, B cell and NK cell histone acetylation antibody and fluorescent combination scheme
Table 5: macrophage histone methylation antibody and fluorescent combination protocol
Table 6: macrophage histone acetylation antibody and fluorescent combination protocol
4. Incubation for 30 min at C in dark, washing twice with staining buffer; followed by pre-fixation with 1% formaldehyde for 15 min, fixation with 70% ethanol for 15 min, washing twice with PBS, permeabilizing the cells with 1 XeBioscience (TM) permeabilization solution for 10 min, adding 1. Mu.L of Mouse Fc Block per well, and incubating at 4℃for 5 min in the absence of light;
then adding intracellular fluorescent antibodies Foxp3 and Di-Methyl-Histone H3 (Lys 9) (D85B 4) XP rabit mAb or actyl-Histone H2B (Lys 5) (D5H 1S) XP Xrabit, incubating for 30 min at 4 ℃ in the absence of light, and washing twice with 1 x eBioscience (TM) permeabilizing solution.
During methylation staining, a secondary antibody Anti-rabit IgG (H+L), F (ab') 2 Fragment was added for 30 min, then 1 XeBioscience (TM) permeabilization solution was washed twice, and then 200. Mu.L staining buffer was used to re-suspend the cells;
cells were resuspended directly after completion of the acetylation staining with 200 μl staining buffer. The sample is transferred to a flow tube for on-line detection.
According to the scheme, methylation and acetylation modification degrees of various immune cell histones are detected. The results of immunocyte grouping are shown in FIG. 1, the results of flow cytometry for detecting the methylation and acetylation modification levels of histones on various immune cells are shown in FIG. 2 and FIG. 3, and the differences of the methylation and acetylation modification levels of histones after stimulation with 900 ng/mL LPS are shown in FIG. 4.
As shown in FIG. 1, the cells are fixed by using 1% formaldehyde and 70% ethanol, the immune cells are normally clustered, and the percentage of the immune cells in the spleen is also in accordance with the report in the literature, for example, about 59.6% of B cells in the spleen and about 37% of T cells, which indicates that the fixing method is stable and reliable and does not influence the cell staining result.
As shown in fig. 2 and 3, the flow cytometry shows different kurtosis when detecting histone methylation and acetylation modification levels on various immune cells, namely, the histone methylation and acetylation modification levels on different immune cells are not consistent, specifically, fig. 2 shows the methylation modification levels of histones on different immune cells, in fig. 2 and 3, isotype control, CD 4T cells, CD 8T cells, treg cells, NK cells, B cells and macrophages are respectively in sequence from top to bottom, and as can be seen from fig. 2, the methylation modification level of macrophage histone is the highest, and in fig. 3, acetylation is the highest modification level on NK cells.
As shown in fig. 4 and 5, before and after stimulation of mice spleen cells with 900 ng/mL LPS, fig. 4 shows B-cell histone methylation modification levels, and fig. 5 shows macrophage histone acetylation modification levels, it can be seen that both B-cell histone methylation modification levels and macrophage histone acetylation modification levels significantly increased, and that after LPS stimulation, macrophage histone acetylation levels tended to be uniform, i.e., changed from original bimodal to nearly unimodal.
In conclusion, the invention can be used for replacing the traditional detection methods with low flux and high cost such as chromatin immunoprecipitation, western blot, immunofluorescence and the like based on flow cytometry, detecting the methylation and acetylation modification levels of the cytohistone, can be specific to the level of a specific cell group, and meets the requirements of scientific researchers for detecting the methylation and acetylation modification levels of the histone on various specific cell groups such as T cells, macrophages or B cells.
In summary, the above embodiments are only preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, but any modifications, equivalent substitutions, improvements, etc. within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (9)
1. A method for detecting histone methylation and acetylation modification levels of a specific cell population based on flow cytometry, which is characterized by taking formaldehyde and ethanol as fixatives, performing flow cytometry detection on the histone methylation and acetylation modification levels of a cell sample to be detected, and comprising the following steps of:
step 1, preparing a fixing agent: preparing 1% formaldehyde as a pre-fixing agent and preparing 70% ethanol as a fixing agent;
step 2, screening a fluorescent channel to be detected: firstly, using BD cell fixing liquid as a fixing agent, detecting the fluorescence intensity and the expression percentage of a CD3 antibody under each fluorescent channel, then fixing a sample by using the fixing agent obtained in the step 1, detecting the fluorescence intensity and the expression percentage of the CD3 antibody under each fluorescent channel, comparing the results, and screening out fluorescent channels which are not influenced by the components of the fixing agent for subsequent detection;
step 3, grouping sample cells according to the expression intensity of immune cell markers, and designing a combination mode of a fluorescent channel and an antibody under different cell populations, wherein the combination mode comprises a methylation detection combination mode and an acetylation detection combination mode;
and 4, performing flow staining on the sample according to the combination mode designed in the step 3, and measuring the histone methylation and acetylation modification level of the sample by adopting a flow cytometry.
2. The method for detecting histone methylation and acetylation modification levels of a specific cell population based on flow cytometry according to claim 1, wherein in step 2, the fluorescent channel to be tested comprises BUV395, BUV496, BUV737, BV421, BV605, BV711, BV786, BV510, FITC, PE, APC and AF700.
3. The method of detecting Histone methylation and acetylation modification levels of a particular cell population based on flow cytometry of claim 1, wherein in step 3, the antibodies comprise CD45, CD3, CD4, CD8, CD11B, CD19, CD25, F4/80, NK1.1, foxp3, and actyl-history H2B (Lys 5) (D5H 1S) XP bar.
4. The method of detecting histone methylation and acetylation modification levels of a particular cell population based on flow cytometry of claim 1, wherein in step 3, the cell population comprises CD 4T cells, CD 8T cells, regulatory T cells (tregs), B cells, NK cells, and macrophages.
5. The method for detecting Histone methylation and acetylation modification levels of a specific cell population based on flow cytometry according to claim 1, wherein a first antibody and a second antibody are further provided in a combination of methylation detection, wherein the first antibody is Di-Methyl-history H3 (Lys 9) (D85B 4) XP or a Rabbit mAb, and the second antibody is Anti-Rabbit IgG (h+l) and F (ab') 2 Fragment.
6. The method for detecting histone methylation and acetylation modification levels of a specific cell population based on flow cytometry according to claim 1, wherein the methylation detection is performed in a combination of T cells, B cells and NK cells in a combination of fluorescent channels and antibodies as follows:
BUV395 fluorescein coupled CD45 antibody, BUV496 fluorescein coupled CD4 antibody, BUV737 fluorescein coupled CD8 antibody, BV605 fluorescein coupled CD19 antibody, BV711 fluorescein coupled CD25 antibody, BV786 fluorescein coupled NK1.1 antibody, FITC fluorescein coupled CD3 antibody, PE fluorescein coupled Foxp3 antibody.
7. The method for detecting histone methylation and acetylation modification levels of a specific cell population based on flow cytometry according to claim 1, wherein the combination of acetylation detection is performed by combining fluorescent channels with antibodies under T cell, B cell and NK cell populations as follows:
BUV395 fluorescein coupled CD45 antibody, BUV496 fluorescein coupled CD4 antibody, BUV737 fluorescein coupled CD8 antibody, BV605 fluorescein coupled CD19 antibody, BV711 fluorescein coupled CD25 antibody, BV786 fluorescein coupled NK1.1 antibody, FITC fluorescein coupled CD3 antibody, APC fluorescein coupled Foxp3 antibody, PE fluorescein coupled Acetyl-Histone H2B (Lys 5) (D5H 1S) XP antibody.
8. The method for detecting histone methylation and acetylation modification levels of a specific cell population based on flow cytometry of claim 1, wherein the methylation detection is combined in a manner such that, under a population of macrophages, a fluorescent channel is combined with an antibody as follows:
BUV395 fluorescein coupled CD45 antibody, BV510 fluorescein coupled F4/80 antibody, BV711 fluorescein coupled CD19 antibody, FITC fluorescein coupled CD3 antibody, AF700 fluorescein coupled CD11b antibody.
9. The method for detecting histone methylation and acetylation modification levels of a specific cell population based on flow cytometry of claim 1, wherein the combination of acetylation detection is performed in a manner such that, under a population of macrophages, the fluorescent channel is combined with an antibody as follows:
BUV395 fluorescein coupled CD45 antibody, BV510 fluorescein coupled F4/80 antibody, BV711 fluorescein coupled CD19 antibody, FITC fluorescein coupled CD3 antibody, AF700 fluorescein coupled CD11B antibody, PE fluorescein coupled Acetyl-Histone H2B (Lys 5) (D5H 1S) XP Rabbit antibody.
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