CN110018143A - The method for detecting dinoflagellate Apoptosis - Google Patents

Abstract

The invention discloses a method for detecting dinoflagellate cell apoptosis. The method for detecting dinoflagellate cell apoptosis includes the following steps: performing apoptosis treatment on the dinoflagellate cells; concentrating the dinoflagellate cells with apoptosis treatment; performing AnnexinV and PI staining processing on the dinoflagellate cell concentrate; The dinoflagellate cell fluid was processed by imaging flow cytometry; the apoptosis rate of dinoflagellate cells was calculated according to AnnexinV/PI scattergram. The method for detecting dinoflagellate cell apoptosis of the present invention can effectively distinguish AnnexinV fluorescence, propidium iodide fluorescence and chlorophyll autofluorescence through imaging flow cytometer, so as to directly quantitatively detect the dinoflagellate cells that have undergone apoptosis in the dinoflagellate cells to be tested. The number of cells can be obtained to obtain the early apoptosis rate of cells, and it also has the advantages of reliable quantification, accurate analysis, and high accuracy of results.

Description

Method for detecting apoptosis of dinoflagellate cells

technical field

The invention belongs to the technical field of cell detection, in particular to a method for detecting dinoflagellate cell apoptosis.

Background technique

Red tide is a harmful ecological phenomenon that causes the water body to change color after a massive outbreak of plankton. There are more than 300 species that can cause red tide, such as the red tide harmful dinoflagellate Alexandrium tamarense, which can release paralytic shellfish poison ( paralyticshell fish poisoning (PSP), shell poisoning poses a serious threat to marine economy and human health after entering the environment. Programmed cell death (PCD) is the autonomous and orderly death of cells regulated by genes during growth or under environmental stress. In order to effectively control red tide, it is of great significance to understand the apoptosis of dinoflagellate cells.

At present, the most widely used methods of apoptosis detection mainly include cell membrane phospholipid serine (PS) detection, Caspase enzyme activity detection, in situ DNA labeling detection method, and ROS detection. Among them, taking the cell membrane phospholipid serine (PS) detection method as an example, the principle is to use AnnexinV/PI double staining to detect cell apoptosis. When cells are in the early stage of apoptosis, phospholipid serine (PS) in the cell membrane will be transferred from the cell membrane to the outside of the cell membrane, making it exposed on the outer surface of the cell membrane, while AnnexinV is a calcium-dependent phospholipid binding protein, which can interact with PS. For specific binding, PI is a nucleic acid dye that cannot penetrate the complete cell membrane, but for cells in the middle and late stages of apoptosis and dead cells, PI can penetrate the cell membrane and stain the nucleus red. Therefore, AnnexinV/PI double staining method can distinguish normal cells, early apoptosis, late apoptosis and necrotic cells.

Flow cytometry is mainly used in medical research such as cell viability and cell identification. In terms of apoptosis and cell signal transduction, the method of detecting apoptotic PS by AnnexinV/PI double staining is widely used. However, this method has limitations in the detection of dinoflagellate cell apoptosis, because dinoflagellate cells have autofluorescence, and ordinary flow cytometry generally cannot distinguish the red fluorescence of dinoflagellate cells from chlorophyll (Chla) and propidium iodide (propidium iodide). The molecular formula of PI is the fluorescence emitted by C ₂₇ H ₃₄ I ₂ N ₄ ), and the appearance characteristics of dinoflagellate cells cannot be visually observed during use, and it is difficult to distinguish some dinoflagellate cells at different characteristic boundaries when analyzing sample data. In addition, it is impossible to accurately locate the fluorescent signal in a single cell, resulting in inaccurate results, and also has the defects of high technical requirements and large sample consumption.

SUMMARY OF THE INVENTION

The object of the present invention is to overcome the deficiencies of the prior art, and to provide a method for detecting the apoptosis of dinoflagellate cells, so as to solve the problem that the existing common flow cytometer cannot distinguish the spontaneous Chla red fluorescence of dinoflagellate cells from the fluorescence emitted by PI, This leads to a technical problem that the accuracy of the apoptosis detection results is not high.

In order to achieve the object of the invention, the present invention provides a method for detecting apoptosis of dinoflagellate cells. The method for detecting dinoflagellate cell apoptosis includes the following steps:

The dinoflagellate liquid to be detected is subjected to apoptosis induction treatment with a cell apoptosis inducer;

Centrifuging and suspending the dinoflagellate liquid treated by the cell apoptosis induction treatment to obtain a dinoflagellate cell concentrate;

After adding AnnexinV/FITC to the dinoflagellate cell concentrate and performing incubation treatment, then adding propidium iodide for dyeing treatment to obtain dyed dinoflagellate cell liquid;

An imaging flow cytometer will be used to analyze the characteristics of the dinoflagellate cells in the dyed dinoflagellate cell solution, to generate an AnnexinV/PI scattergram, and to select a cell population according to the scattergram, and then according to the selected cell population. The number of dinoflagellate apoptotic cells in the tested dinoflagellate sample is obtained, and the apoptotic rate of dinoflagellate cells is calculated according to the number of dinoflagellate apoptotic cells.

Compared with the prior art, the method for detecting dinoflagellate cell apoptosis of the present invention utilizes phosphatidylserine dyes and PI dyes to dye dinoflagellate cells. The instrument can effectively distinguish AnnexinV fluorescence and propidium iodide fluorescence chlorophyll autofluorescence, so as to directly and quantitatively measure the number of apoptotic dinoflagellate cells in the dinoflagellate cells to be tested, so as to obtain the early apoptosis rate of cells, and it also has quantitative results. Reliable, accurate analysis, high accuracy of results.

Description of drawings

1 is a schematic diagram of the process flow of the method for detecting dinoflagellate cell apoptosis according to an embodiment of the present invention;

Figure 2 is the AnnexinV/PI scatter plot of the dinoflagellate cells in the control group at different stages in Example 1 of the present invention; wherein, Figure 2A is the AnnexinV/PI scatter plot of the dinoflagellate cells in the control group at 0 min, and Figure 2B is the control AnnexinV/PI scatter plot of group dinoflagellate cells at 120min;

3 is the AnnexinV/PI scatter plot of the dinoflagellate cells of the experimental group at different periods in Example 1 of the present invention; wherein, FIG. 3A is the AnnexinV/PI scatter plot of the dinoflagellate cells of the experimental group at 0 min, and FIG. 3B is the experiment AnnexinV/PI scatter plot of group dinoflagellate cells at 120min;

4 is an image of a single dinoflagellate cell in the AnnexinV/PI scatter plot in Example 1 of the present invention;

Figure 5 is the AnnexinV/PI scatter plot of the dinoflagellate cells in the control group at different stages in Example 2 of the present invention; wherein, Figure 5A is the AnnexinV/PI scatter plot of the dinoflagellate cells in the control group at 0 min, and Figure 5B is the control The AnnexinV/PI scatter plot of group dinoflagellate cells at 60min;

Figure 6 is the AnnexinV/PI scatter plot of the dinoflagellate cells of the experimental group at different stages in Example 2 of the present invention; wherein, Figure 6A is the AnnexinV/PI scatter plot of the dinoflagellate cells of the experimental group at 0 min, and Figure 6B is the experiment AnnexinV/PI scatter plot of group dinoflagellate cells at 60min.

Detailed ways

In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer, the present invention will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, but not to limit the present invention.

In the embodiment of the present invention, the following terms are described as follows.

Phosphatidylserine: (Phosphatidlserinea, PS) It is normally located on the inner side of the cell membrane, but in the early stage of apoptosis, PS can be flipped from the inner side of the cell membrane to the surface of the cell membrane and exposed to the extracellular environment.

Annexin V: It is a Ca2 ⁺ -dependent phospholipid binding protein with a molecular weight of 35-36kD, which can specifically bind to PS with high affinity. The Annexin V is labeled with fluorescein (FITC), and the labeled Annexin V is used as a fluorescent probe to detect the occurrence of apoptosis by flow cytometry or fluorescence microscope.

Propidine iodide (PI): It is a nucleic acid dye that cannot penetrate the complete cell membrane, but in the middle and late stages of apoptosis and dead cells, PI can penetrate the cell membrane and stain the nucleus red. Therefore, when Annexin V is used in combination with PI, cells in the early and late stages of apoptosis can be distinguished from dead cells.

The embodiment of the present invention provides a method for detecting apoptosis of dinoflagellate cells. The method for detecting dinoflagellate cell apoptosis includes the following steps:

S01. Apoptosis treatment of dinoflagellate cells: the dinoflagellate liquid to be detected is subjected to apoptosis induction treatment with a cell apoptosis inducer;

S02. Perform a centrifugal suspension treatment on the apoptosis-treated dinoflagellate cells: perform a centrifugal suspension treatment on the dinoflagellate liquid treated by the apoptosis induction to obtain a dinoflagellate cell concentrate;

S03. Perform AnnexinV and PI staining treatment on the dinoflagellate cell concentrate: add AnnexinV/FITC to the dinoflagellate cell concentrate and incubate, and then continue to add propidium iodide for dyeing to obtain dyed dinoflagellate cells liquid;

S04. Perform imaging flow detection processing on the dyed dinoflagellate cell solution: use an imaging flow cytometer to perform flow detection on the dinoflagellate cells in the dyed dinoflagellate cell solution to generate volume/aspect ratio scatter points of the dinoflagellate cells Figure to obtain the required dinoflagellate cell population; then generate AnnexinV/PI scatter plot according to the selected dinoflagellate cell population;

S05. Calculate the apoptotic rate of dinoflagellate cells according to the AnnexinV/PI scattergram: according to the AnnexinV/PI scattergram, obtain the number of dinoflagellate apoptotic cells in the dinoflagellate liquid to be detected, according to the The number of dinoflagellate apoptotic cells was used to calculate the dinoflagellate cell apoptosis rate in the dinoflagellate liquid to be detected.

Wherein, in the step S01, after the apoptosis-inducing agent is added to the dinoflagellate liquid to be detected, the apoptosis-inducing agent will act to induce the occurrence of dinoflagellate cells in the dinoflagellate liquid to be detected. apoptosis. Wherein, the apoptosis-inducing agent can be selected from different apoptosis-inducing agents as required. In one embodiment, the apoptosis-inducing agent may include at least one of dinoflagellate, H ₂ O ₂ and virus. Among them, the dinoflagellate bacterium (6A1) was isolated by our research group from the red tide seawater that broke out in the Dongshan sea area of Dapeng Bay, Shenzhen. Of course, the dinoflagellate bacteria can also be other alginolytic bacteria. Of course, the apoptosis of the dinoflagellate cells can also be achieved by constructing the environment of cell apoptosis, such as constructing nitrogen starvation, iron starvation, etc. to achieve the apoptosis of the dinoflagellate cells.

In addition, the selected types of the apoptosis-inducing agent are different, and the amount added to the dinoflagellate liquid to be detected is different, but generally the amount of the apoptosis-inducing agent added to the dinoflagellate liquid to be detected is different. The amount does not exceed 10% of the volume ratio of the dinoflagellate liquid to be detected. In one embodiment, the volume ratio of the apoptosis-inducing agent added to the dinoflagellate solution to be detected is 1%-10%.

For the accuracy of detection, in one embodiment, the dinoflagellate liquid to be detected is divided into several parts, and the several parts of the dinoflagellate liquid to be detected are divided into an experimental group and a blank control group; wherein, the experimental group is A certain amount of the apoptosis-inducing agent is added to the dinoflagellate liquid to be detected, and further, when the 6A1 is selected as the apoptosis-inducing agent, because 6A1 contains its own culture medium, such as containing autoclave. Bacteria 2216E medium, in order to verify the effect of the 6A1-containing medium on dinoflagellate cell apoptosis, the experimental group was to add a certain amount of the 6A1 to the dinoflagellate liquid to be detected, and the blank The control group is to add the described 6A1 substratum of the same amount as the experimental group in the dinoflagellate liquid to be detected (that is, do not add the inhibitory bacteria contained in 6A1, only add the substratum contained in 6A1, such as autoclaving. 2216E medium of bacteria).

Of course, the experimental group and the blank control group were placed under the same conditions for culture treatment after the setting was completed.

In order to further improve the detection accuracy, each group of the experimental group and the blank control group is set with 2-4 parallel samples, and the dinoflagellate cell content in each of the dinoflagellate liquids to be detected is controlled the same to ensure the detection accuracy. Comparability. In an embodiment, the dinoflagellate cell content in each piece of the dinoflagellate liquid to be detected is 10 ⁵ -10 ⁶ cells/ml; in a specific embodiment, the dinoflagellate in the dinoflagellate liquid to be detected can be Not just for Tama Alexander algae. By controlling the content of dinoflagellate cells in the dinoflagellate liquid to be detected and the number of parallel samples, the detection accuracy is improved. In addition, the medium of the dinoflagellate liquid to be detected can be but not only autoclaved f/2 medium.

In the step S02, the centrifugation and suspension treatment of the dinoflagellate liquid subjected to the apoptosis induction treatment can be carried out according to the following method:

The dinoflagellate liquid subjected to the apoptosis-inducing treatment is centrifuged, the precipitated dinoflagellate cells are collected, and then the precipitated dinoflagellate cells are subjected to suspension treatment. For suspension treatment solution can be used but not only deionized water to dilute Binding Buffer (AnnexinV/FITC Binding Buffer) 1:3. In one embodiment, the concentration of dinoflagellate cells in the dinoflagellate cell concentrate obtained may be controlled at 10 ⁵ -10 ⁶ cells/ml, specifically 10 ⁵ cells/ml.

In the step S03, AnnexinV/FITC is added to the dinoflagellate cell concentrate to achieve staining of the dinoflagellate cells. In one embodiment, the AnnexinV/FITC is added per 100 μl of the dinoflagellate cell concentrate. The ratio of 1-5 μl of the AnnexinV/FITC is added to the dinoflagellate cell concentrate, wherein the dinoflagellate cell concentration in the dinoflagellate cell concentrate is 10 ⁵ cells/ml. In addition, adding AnnexinV/FITC to realize the dyeing and incubation process of dinoflagellate cells, the incubation process can be performed at room temperature in the dark, such as incubation for 5 min.

After AnnexinV/FITC completes the dyeing treatment of the dinoflagellate cell concentrate, then add propidium iodide to the dinoflagellate cell concentrate for dyeing treatment. The ratio of adding 0.1-0.5 μg of the propidium iodide per 100 μl of the dinoflagellate cell concentrate is added to the dinoflagellate cell concentrate, wherein the dinoflagellate cell concentration in the dinoflagellate cell concentrate is 10 ⁵ cells/ml. Control the dyeing degree of dinoflagellate cells by controlling the amount of dye added, thereby controlling the regulation of the dyeing effect of dinoflagellate cells, thereby improving the accuracy of the final dinoflagellate cell apoptosis rate detection.

In the step S04, the method for generating the volume/aspect ratio scatterplot of the dinoflagellate cells includes the following steps:

Take the dyed dinoflagellate cell solution described in step S03 and put it on the imaging flow cytometer, then adjust the laser power of the imaging flow cytometer to the upper power limit, and then adjust the laser power to gradually reduce until the fluorescence is avoided Saturate and then generate the volume/aspect ratio scatterplot.

Wherein, the power of the laser controlling the imaging flow cytometer is gradually reduced from the upper limit until fluorescence saturation is avoided, in order to obtain a clear and high-quality volume/aspect ratio scattergram (Area/Aspect Ratio scattergram). In one embodiment, the laser power is gradually reduced from the upper limit until the laser power is 4-20 MV when the fluorescence saturation is avoided. At this time, the original maximum pixel of the generated volume/aspect ratio scattergram ( raw max pixel)<4096. Clear and high quality volume/aspect ratio scatterplots were obtained by adjusting the laser power to 4-20 MV. Among them, fluorescence saturation means that in general, the substance will fluoresce when excited, and the intensity of the fluorescence will increase with the increase of the excitation intensity, but when the excitation intensity reaches a certain level, the intensity of the fluorescence will begin to tend to A constant value, instead of increasing due to the increase of excitation intensity, we say that fluorescence saturation occurs.

On the basis of adjusting the laser power of the imaging flow cytometer, the flow rate of the dyed dinoflagellate cell solution in the imaging flow cytometer can also be reduced to increase the fluorescence intensity of the instrument for each channel. Sensitivity, as in a further embodiment, the flow rate at which the dyed dinoflagellate cell fluid is injected in the imaging flow cytometer is controlled to be 1 μl/min-4 μl/min.

Therefore, in a specific embodiment, when the dinoflagellate in the dinoflagellate liquid to be detected is dinoflagellate, and according to the volume/aspect ratio scattergram generated in step S04, the aspect ratio is selected in the range of 0-0.6 of dinoflagellate cell populations. When the dinoflagellate in the dinoflagellate liquid to be detected is other, all the dinoflagellate cell populations in the volume/aspect ratio scattergram generated in step S04 may be selected.

In one embodiment, the method for generating the AnnexinV/PI scatterplot according to the dinoflagellate cell population selected in step S04 comprises the following steps:

After performing fluorescence compensation processing on the dinoflagellate cell population by using the analysis software of the imaging flow cytometer, the AnnexinV/PI cell scatterplot is generated.

Wherein, the method for performing the fluorescence compensation on the dinoflagellate cell population by using the analysis software of the imaging flow cytometer comprises the following steps:

Obtain chlorophyll single fluorescence sample data to generate a single fluorescence sample data file, select the Compensation window in the analysis software to import the single fluorescence sample data file and automatically generate a compensation matrix (CompensationMatrix), and adjust the red matrix (Matrix) After the image verification is completed, the compensation file is completed, and the compensation file is imported when analyzing the data to realize the fluorescence compensation of the dinoflagellate cell population.

In a specific embodiment, in generating the AnnexinV/PI cell scatterplot, AnnexinV fluorescence is the second channel, PI fluorescence is the fourth channel, and the autofluorescence of chlorophyll contained in dinoflagellate cells is in the fifth channel. In the generated AnnexinV/PI cell scatterplot, its abscissa is Intensity_MC_AnnexinV, and its ordinate is Intensity_MC_PI. Therefore, the AnnexinV/PI cell scattergram can be verified by the images of the dinoflagellate cells to divide the gate into four subgroups, namely normal viable cell subgroup, early apoptotic cell subgroup, and late apoptotic cell subgroup. Cell subsets, necrotic cell subsets.

In addition, the imaging flow cytometer in the step S05 may but not only be FlowSight, the analysis software can be analysis software ideas and the like.

In the step S05, since the AnnexinV/PI scattergram generated in the step S05, the AnnexinV fluorescence, the PI fluorescence and the chlorophyll autofluorescence contained in the dinoflagellate cells are displayed in different fifth channels, respectively. The apoptosis method can effectively distinguish AnnexinV fluorescence and propidium iodide fluorescence chlorophyll autofluorescence, so as to directly and quantitatively measure the number of apoptotic dinoflagellate cells in the tested dinoflagellate cells, thereby obtaining the early cell apoptosis rate. In one embodiment, calculating the dinoflagellate cell apoptosis rate in the dinoflagellate liquid to be detected according to the number of the dinoflagellate apoptotic cells is calculated according to the following formula:

The dinoflagellate cell apoptosis rate %=C _X /C ₀ ×100%

In the formula, C _X is the number of apoptotic cells in the xth dinoflagellate solution to be detected; C ₀ is the total number of dinoflagellate cells in the xth dinoflagellate solution to be detected collected by the imaging flow cytometer.

Therefore, the method for detecting dinoflagellate cell apoptosis described above uses AnnexinV/FITC and PI dyes to stain dinoflagellate cells. According to the characteristics of dinoflagellate cells and apoptosis characteristics, AnnexinV can be effectively distinguished by imaging flow cytometry Fluorescence, propidium iodide fluorescence chlorophyll autofluorescence, so as to directly quantitatively measure the number of dinoflagellate cells that have undergone apoptosis in the dinoflagellate cells to be tested, thereby obtaining the early apoptosis rate of cells, and also has quantitative and reliable, accurate analysis, The result has the advantage of high precision. Effectively overcome the existing AnnexinV/PI double staining method, which cannot effectively distinguish the spontaneous Chla red fluorescence of dinoflagellate cells from the fluorescence emitted by PI, and cannot visually observe the appearance characteristics of dinoflagellate cells, resulting in difficulty in distinguishing the parts at different feature boundaries. Dinoflagellate cell populations and the inability to precisely locate fluorescent signals within individual cells.

The present invention will now be described in further detail with reference to specific examples.

Example 1

This embodiment provides a method for detecting apoptosis of dinoflagellate cells. The method for detecting dinoflagellate cell apoptosis includes the following steps:

S11: Take 4 volumetric flasks and add Tama Alexandria dinoflagellate solution (the medium of Tama Alexandria dinoflagellate solution is 2216E medium), and divide the 4 volumetric flasks into 2 groups: experimental group (2 bottles), blank The control group (2 bottles); and the number of dinoflagellate cells in the experimental group and the blank control group was the same; wherein, 5% 6A1 by volume was added to the volumetric flask of the experimental group (wherein, 6A1 contained 10 ⁸ cells/ ml of inhibitory cells, the culture medium is 2216E medium); add 5% 6A1 2216E medium by volume to the volumetric flask of the control group (that is, the control group does not contain 6A1 inhibitory cells); The supernatant was induced under the same conditions as the control group for 120 min;

S12: Centrifuge the dinoflagellate cells of each group treated in step S11 at 1000 rpm for 5 min, precipitate the cells, remove the supernatant, add about 1 ml of PBS pre-cooled at 4°C, resuspend the cells, and centrifuge again to precipitate the cells, and remove the supernatant by suction. clear; dilute the binding buffer (4ml 4* binding buffer+12ml deionized water) with deionized water at 1:3, resuspend the cells with 1* binding buffer, and obtain the dinoflagellate cell concentrate;

S13: Take 100 μl of the dinoflagellate cell concentrate (the dinoflagellate cell concentration is 2×10 ⁵ cells/ml), add 5 μl of AnnexinV/FITC, mix well, incubate at room temperature in the dark, and add 10 μl of 20ug/ml after 5 minutes The propidium iodide solution (PI) was obtained to obtain dyed dinoflagellate cell fluid;

S14: Check on the machine immediately after sampling, adjust the laser power to 4MV, and inject the dyed dinoflagellate cell solution into the imaging flow cytometer at a flow rate of 3 μl/min, generate an Area/Aspect Ratio scatter plot, and obtain The cell population with Area between 500-2500μm ³ and Aspect Ratio between 0-0.6;

S15: Utilize FlowSight's analysis software ideas first perform fluorescence compensation on the cell population in step S13, and then generate a scatter plot of AnnexinV/PI cells. The analysis and comparison in the figure shows the area of apoptosis of Alexandrina tamarina cells. The result is AnnexinV/PI. PI scatter plot; among them, the AnnexinV/PI scatter plot of the control group is shown in Figure 2, and the AnnexinV/PI scatter plot of the experimental group is shown in Figure 3;

S16: According to the calculation formula of the dinoflagellate cell apoptosis rate from Figure 2 and Figure 3, it can be calculated that the early apoptosis rate of the control group is 0% at 0min, and the early apoptosis rate is 3.21% at 120min. The early withering rate of the group was 0% at 0min, and 47.59% at 120min.

And from Figure 2 and Figure 3, the upper left area Q1 is AnnexinV-/PI+, indicating necrotic cells; the upper right area Q2 is AnnexinV+/PI+, indicating late apoptotic cells; the lower left area Q3 is AnnexinV-/PI-, indicating normal Live cells; Q4 in the lower right corner is AnnexinV+/PI-, indicating early apoptotic cells. Further images of individual dinoflagellate cells in different states, each cell has four images, BF (bright field), AnnexinV (false green), PI (false orange), Chla (false red) and BF/PI/ AnnexinV, specifically shown in Figure 4.

Example 2

This embodiment provides a method for detecting apoptosis of dinoflagellate cells. The method for detecting dinoflagellate cell apoptosis includes the following steps:

S11: Take 4 volumetric flasks and add Tama Alexandrina dinoflagellate liquid (the medium of Tama Alexandria dinoflagellate liquid is 2216E medium), and divide the 4 volumetric flasks into 2 groups: experimental group (2 bottles), control group group (2 flasks); and the number of dinoflagellate cells in the experimental group and the blank control group was the same; wherein, 10% volume of 6A1 was added to the volumetric flask of the experimental group (wherein, 6A1 contained 10 ⁸ cells/ml Bacteriostatic cells, the medium is 2216E medium); add 10% volume of 6A1 2216E medium to the volumetric flask of the control group (that is, the control group does not contain 6A1 inhibitory cells); the experimental group and The control group was subjected to supernatant induction for 60 min under the same conditions;

S12: Centrifuge the dinoflagellate cells of each group treated in step S11 at 1000 rpm for 5 min, precipitate the cells, remove the supernatant, add about 1 ml of PBS pre-cooled at 4°C, resuspend the cells, and centrifuge again to precipitate the cells, and remove the supernatant by suction. clear; dilute the binding buffer (4ml 4* binding buffer+12ml deionized water) with deionized water at 1:3, resuspend the cells with 1* binding buffer, and obtain the dinoflagellate cell concentrate;

S13: Take 100 μl of the dinoflagellate cell concentrate (the dinoflagellate cell concentration is 2×10 ⁵ cells/ml), add 1 μl of AnnexinV/FITC, mix well, incubate at room temperature in the dark, and add 20 μl of 20ug/ml after 5 minutes The propidium iodide solution (PI) was obtained to obtain dyed dinoflagellate cell fluid;

S14: Check on the machine immediately after sampling, adjust the laser power to 4MV, and inject the dyed dinoflagellate cell solution into the imaging flow cytometer at a flow rate of 4 μl/min, generate an Area/Aspect Ratio scatter plot, and obtain The cell population with Area between 500-2500μm ³ and Aspect Ratio between 0-0.6;

S15: Utilize FlowSight's analysis software ideas first perform fluorescence compensation on the cell population in step S13, and then generate a scatter plot of AnnexinV/PI cells. The analysis and comparison in the figure shows the area of apoptosis of Alexandrina tamarina cells. The result is AnnexinV/PI. PI scatter plot; among them, the AnnexinV/PI scatter plot of the control group is shown in Figure 5, and the AnnexinV/PI scatter plot of the experimental group is shown in Figure 6;

S16: According to the calculation formula of the above-mentioned dinoflagellate cell apoptosis rate from Figure 2 and Figure 3, it can be calculated that the early apoptosis rate of the control group is 0% at 0 min, and the early apoptosis rate is 0.37% at 60 min. The early withering rate was 0% at 0min and 63.6% at 60min.

To sum up, the methods for detecting dinoflagellate cell apoptosis in the above examples can be seen, the method for detecting dinoflagellate cell apoptosis provided by the embodiments of the present invention can effectively distinguish AnnexinV fluorescence and propidium iodide fluorescence chlorophyll autofluorescence, so as to directly quantitatively detect The number of apoptotic dinoflagellate cells in the dinoflagellate cells to be measured can be used to obtain the early apoptosis rate of the cells, and it also has the advantages of reliable quantification, accurate analysis and high result accuracy. Therefore, it is more convenient, faster and more accurate than the traditional detection method.

The above descriptions are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention shall be included in the protection of the present invention. within the range.

Claims (10)

1. a method for detecting dinoflagellate cell apoptosis, is characterized in that, comprises the steps: The dinoflagellate liquid to be detected is subjected to apoptosis induction treatment with a cell apoptosis inducer; Centrifuging and suspending the dinoflagellate liquid treated by the apoptosis induction to obtain a dinoflagellate cell concentrate; After adding AnnexinV/FITC to the dinoflagellate cell concentrate and performing incubation treatment, then adding propidium iodide for dyeing treatment to obtain dyed dinoflagellate cell liquid; The dinoflagellate cells in the dyed dinoflagellate cell solution were detected by flow cytometry, and the volume/aspect ratio scatter plot of the dinoflagellate cells was generated to obtain the desired dinoflagellate cell population; The dinoflagellate cell population generates an AnnexinV/PI scatterplot; According to the AnnexinV/PI scattergram, the number of dinoflagellate apoptotic cells in the dinoflagellate liquid to be detected is obtained, and the number of dinoflagellate apoptotic cells in the dinoflagellate liquid to be detected is calculated according to the number of dinoflagellate cells to be detected. Apoptosis rate of dinoflagellate cells. 2. The method according to claim 1, wherein the method for generating the volume/aspect ratio scatterplot of the dinoflagellate cells comprises the steps of: Take the dyed dinoflagellate cell solution and put it on the imaging flow cytometer, then adjust the laser power of the imaging flow cytometer to the upper power limit, and then adjust the laser power to gradually reduce until the fluorescence saturation is avoided, and then The volume/aspect ratio scatterplot is generated. 3. The method according to claim 2, wherein the laser power is gradually reduced until the laser power is 4-20 MV when the laser power is gradually reduced until the fluorescence saturation is avoided; The injection flow rate of the dyed dinoflagellate cell solution in the imaging flow cytometer is 1 μl/min-4 μl/min. 4. The method according to claim 2 or 3, wherein the dinoflagellate cell population with an aspect ratio of 0-0.6 is selected according to the volume/aspect ratio scatterplot. 5. method according to claim 1 is characterized in that: the method for generating described AnnexinV/PI scatterplot according to the described dinoflagellate cell population selected comprises the steps: After performing fluorescence compensation processing on the dinoflagellate cell population by using the analysis software of the imaging flow cytometer, the AnnexinV/PI cell scatterplot is generated. 6. The method according to claim 5, wherein the method for performing the fluorescence compensation on the dinoflagellate cell population by utilizing the analysis software of the imaging flow cytometer comprises the following steps: Obtain chlorophyll single fluorescence sample data to generate a single fluorescence sample data file, select the compensation window in the analysis software to import the single fluorescence sample data file and automatically generate a compensation matrix, adjust the matrix value marked in red, and complete the compensation file after image verification , and importing the compensation file when analyzing data to perform fluorescence compensation on the dinoflagellate cell population. 7. The method according to claim 5 or 6, wherein in generating the AnnexinV/PI cell scatterplot, AnnexinV fluorescence is the second channel, PI fluorescence is the fourth channel, and dinoflagellate cells contain Chlorophyll autofluorescence in the fifth channel. 8. The method according to any one of claims 1-3, 5-6, wherein the dinoflagellate apoptosis in the dinoflagellate liquid to be detected is calculated according to the number of the dinoflagellate apoptotic cells The rate is calculated according to the following formula: The dinoflagellate cell apoptosis rate %=C _X /C ₀ ×100%; In the formula, C _X is the number of apoptotic cells in the xth dinoflagellate solution to be detected; C ₀ is the total number of dinoflagellate cells in the xth dinoflagellate solution to be detected collected by the imaging flow cytometer. 9. The method according to any one of claims 1-3 and 5-6, wherein the dinoflagellate in the dinoflagellate liquid to be detected is Alexandrium tamarina. 10. The method according to any one of claims 1-3, 5-6, characterized in that: the dinoflagellate cell concentration in the dinoflagellate cell concentrate is 10 ⁵ -10 ⁶ cells/ml; and/or The AnnexinV/FITC is added to the dinoflagellate cell concentrate according to the ratio of 1-5 μl of the AnnexinV/FITC per 100 μl of the dinoflagellate cell concentrate, wherein the formazan in the dinoflagellate cell concentrate is added. Algal cells at a concentration of 10 ⁵ -10 ⁶ cells/ml; and/or The propidium iodide is added to the dinoflagellate cell concentrate in a ratio of 0.1-0.5 μg of the propidium iodide per 100 μl of the dinoflagellate cell concentrate, wherein the dinoflagellate cell concentrate is The dinoflagellate cell concentration was 10 ⁵ -10 ⁶ cells/ml.