CN110018143B - Method for detecting dinoflagellate apoptosis - Google Patents
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Abstract
本发明公开了一种检测甲藻细胞凋亡的方法。所述检测甲藻细胞凋亡的方法包括步骤有：对甲藻细胞进行凋亡处理；对凋亡处理的甲藻细胞进行浓缩处理；对甲藻细胞浓缩液进行AnnexinV、PI染色处理；对染色甲藻细胞液进行成像流式检测处理；依据AnnexinV/PI散点图计算甲藻细胞的凋亡率。本发明检测甲藻细胞凋亡的方法通过成像流式细胞仪能够有效区分AnnexinV荧光、碘化丙啶荧光、叶绿素自发荧光，从而直接定量测出待测甲藻细胞中发生凋亡的甲藻细胞个数，从而得出细胞早期凋亡率，而且还具有定量可靠，分析准确，结果精度高等优点。
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
Technical Field
The invention belongs to the technical field of cell detection, and particularly relates to a method for detecting dinoflagellate apoptosis.
Background
Red tide is a harmful ecological phenomenon that water changes color after plankton outbreaks, and more than 300 kinds of red tide can be caused, for example, red tide harmful dinoflagellate Alexandrium tamarense (Alexandrium tamarense) can release Paralytic Shellfish Poisoning (PSP), and the shellfish poisoning poses serious threats to marine economy and human health after entering the environment. Programmed Cell Death (PCD) is autonomous and orderly death regulated by a gene during the growth of cells or under environmental stress, and has a significant meaning in order to effectively control red tide and understand the apoptosis of red tide dinoflagellate cells.
At present, cell membrane phosphoryl lipid serine (PS) detection, Caspase enzyme activity detection, in-situ DNA labeling detection and ROS detection are mainly applied to cell apoptosis detection methods. In the method, the cell membrane Phospholipide Serine (PS) detection method is taken as an example, and the principle is to detect the apoptosis by annexin V/PI double staining. In early apoptosis, phosphoryl lipid serine (PS) in the cell membrane is transferred from inside to outside of the cell membrane to expose it on the outer surface of the cell membrane, annexin V is a calcium-dependent phospholipid binding protein which specifically binds to PS, and PI is a nucleic acid dye which cannot permeate the whole cell membrane, but can permeate the cell membrane to dye the nucleus red for cells in late apoptosis and dead cells. Therefore, normal cells, early apoptosis, late apoptosis and necrotic cells can be distinguished by adopting an annexin V/PI double staining method.
The flow cytometer is mainly used in medical research such as cell viability and cell identification. The method for detecting the cell apoptosis PS by annexin V/PI double staining has wide application in the aspects of cell apoptosis, cell signal transduction and the like. However, the method has a limitation when being applied to detecting the apoptosis of the dinoflagellate cells, because the dinoflagellate cells have autofluorescence, and a common flow cytometer can not distinguish the spontaneous chlorophyll (Chla) red fluorescence of the dinoflagellate cells from the propidium iodide (the PI molecular formula is C)27H34I2N4) The emitted fluorescence cannot visually observe the appearance characteristics of the dinoflagellate cells while in use, partial dinoflagellate cell populations in different characteristic limits are difficult to distinguish when sample data are analyzed, and a fluorescence signal in a single cell cannot be accurately positioned, so that the result accuracy is not high, and the defects of high technical requirements and large sample consumption are also existed.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for detecting dinoflagellate apoptosis so as to solve the technical problem that the accuracy of an apoptosis detection result is low because the conventional common flow cytometer cannot distinguish the spontaneous Chla red fluorescence of dinoflagellate cells from the fluorescence emitted by PI.
In order to achieve the purpose, the invention provides a method for detecting dinoflagellate apoptosis. The method for detecting the dinoflagellate apoptosis comprises the following steps:
carrying out apoptosis induction treatment on the dinoflagellate liquid to be detected by adopting an apoptosis inducer;
performing centrifugal suspension treatment on the dinoflagellate solution subjected to the apoptosis induction treatment to obtain a dinoflagellate cell concentrated solution;
adding annexin V/FITC into the dinoflagellate cell concentrated solution, performing incubation treatment, and then continuously adding propidium iodide for dyeing treatment to obtain dyed dinoflagellate cell solution;
analyzing the characteristics of the dinoflagellate cells in the stained dinoflagellate cell sap by using an imaging flow cytometer to generate an annexin V/PI scattergram, selecting a cell population according to the scattergram, obtaining the number of dinoflagellate apoptotic cells in a detected dinoflagellate sample according to the selected cell population, and calculating the apoptosis rate of the dinoflagellate cells according to the number of dinoflagellate apoptotic cells.
Compared with the prior art, the method for detecting the dinoflagellate apoptosis of the invention dyes the dinoflagellate cells by using the phosphatidylserine dye and the PI dye, and can effectively distinguish annexinV fluorescence and propidium iodide fluorescent chlorophyll autofluorescence through an imaging flow cytometer according to the characteristics and apoptosis characteristics of the dinoflagellate cells, thereby directly and quantitatively detecting the number of the dinoflagellate cells to be detected which are apoptotic, and further obtaining the early apoptosis rate of the cells.
Drawings
FIG. 1 is a schematic process flow diagram of a method for detecting dinoflagellate apoptosis in accordance with an embodiment of the present invention;
FIG. 2 is a dot-plot of AnnexinV/PI cells of control dinoflagellate cells at different times in example 1 of the present invention; wherein, FIG. 2A is AnnexinV/PI scattergram of control group dinoflagellate cells at 0min, and FIG. 2B is AnnexinV/PI scattergram of control group dinoflagellate cells at 120 min;
FIG. 3 is a dot-plot of AnnexinV/PI cells of the experimental group dinoflagellate cells at different times in example 1; wherein, FIG. 3A is AnnexinV/PI scattergram of the experimental group dinoflagellate cells at 0min, and FIG. 3B is AnnexinV/PI scattergram of the experimental group dinoflagellate cells at 120 min;
FIG. 4 is an image of a single dinoflagellate cell in the annexin V/PI scattergram in example 1 of the present invention;
FIG. 5 is a dot-plot of AnnexinV/PI cells of control dinoflagellate cells at different times in example 2 of the present invention; wherein, FIG. 5A is AnnexinV/PI scattergram of control group dinoflagellate cells at 0min, and FIG. 5B is AnnexinV/PI scattergram of control group dinoflagellate cells at 60 min;
FIG. 6 is a dot-plot of AnnexinV/PI cells of the experimental group dinoflagellate cells at different times in example 2; wherein, FIG. 6A is AnnexinV/PI scattergram of the experimental group dinoflagellate cells at 0min, and FIG. 6B is AnnexinV/PI scattergram of the experimental group dinoflagellate cells at 60 min.
Detailed Description
In order to make the technical problems, technical solutions and advantageous effects to be solved by the present invention more clearly apparent, the present invention is further described in detail below with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
In the embodiments of the present invention, the following terms are explained below.
Phosphotalilserine (PS) is normally located inside the cell membrane, but in the early stages of apoptosis, PS can roll over from the inside of the cell membrane to the surface of the cell membrane, exposing it to the extracellular environment.
Annexin V: is Ca with molecular weight of 35-36kD2+The dependent phospholipid binding protein can be specifically combined with PS with high affinity. Annexin V is labeled by Fluorescein (FITC), and the labeled Annexin V is used as a fluorescent probe, and the occurrence of apoptosis can be detected by a flow cytometer or a fluorescence microscope.
Propidium Iodide (PI): is a nucleic acid dye that is impermeable to the intact cell membrane, but in cells in the middle and late stages of apoptosis and dead cells, PI is able to permeate the cell membrane to red stain the nucleus. Therefore, by matching Annexin V with PI, cells with early and late apoptosis can be distinguished from dead cells.
The embodiment of the invention provides a method for detecting dinoflagellate apoptosis. The method for detecting the dinoflagellate apoptosis comprises the following steps:
s01, carrying out apoptosis treatment on dinoflagellate cells: carrying out apoptosis induction treatment on the dinoflagellate liquid to be detected by adopting an apoptosis inducer;
s02, carrying out centrifugal suspension treatment on the dinoflagellate cells subjected to apoptosis treatment: carrying out centrifugal suspension treatment on the dinoflagellate solution subjected to apoptosis induction treatment to obtain a dinoflagellate cell concentrated solution;
s03, performing annexin V and PI staining treatment on the dinoflagellate cell concentrated solution: adding annexin V/FITC into the dinoflagellate cell concentrated solution, performing incubation treatment, and then continuously adding propidium iodide for dyeing treatment to obtain dyed dinoflagellate cell solution;
s04, performing imaging flow detection treatment on the dinoflagellate staining cell sap: performing flow detection on the dinoflagellate cells in the stained dinoflagellate cell sap by using an imaging flow cytometer to generate a volume/aspect ratio scatter diagram of the dinoflagellate cells so as to obtain a required dinoflagellate cell population; generating an annexin V/PI scattergram according to the selected dinoflagellate cell population;
s05, calculating the apoptosis rate of the dinoflagellate cells according to the annexinV/PI scatter diagram: and obtaining the number of the dinoflagellate apoptotic cells in the to-be-detected dinoflagellate liquid according to the annexinV/PI scatter diagram, and calculating the dinoflagellate apoptosis rate in the to-be-detected dinoflagellate liquid according to the number of the dinoflagellate apoptotic cells.
In step S01, when the apoptosis inducing agent is added to the to-be-detected chlorella solution, the apoptosis inducing agent acts to induce chlorella cells in the to-be-detected chlorella solution to undergo apoptosis. Wherein, the apoptosis inducer can be different apoptosis inducers according to the requirement. In one embodiment, the apoptosis-inducing agent may comprise a bacterium selected from the group consisting of M.lysozyme, H2O2And a virus. Wherein the dinoflagellate bacteria (6A1) were isolated from the issue group in the red tide waters of the Shenzhen Dapeng Bay east mountain sea area outbreak. Of course, the dinoflagellate bacteria can also be other dinoflagellate bacteria. Of course, the apoptosis of the dinoflagellate cell can also be realized by constructing an environment of apoptosis of the dinoflagellate cell, such as nitrogen starvation, iron starvation and the like.
In addition, the types of the apoptosis inducers are different, and the apoptosis inducers are added into the to-be-detected alga solution in different amounts, but the amount of the apoptosis inducers added into the to-be-detected alga solution is not more than 10% of the to-be-detected alga solution in volume ratio. As in one embodiment, the apoptosis-inducing agent is added to the solution of the to-be-detected alga at a volume ratio of 1% to 10%.
For the accuracy of detection, in one embodiment, the to-be-detected chlorella solution is divided into a plurality of parts, and the plurality of parts of the to-be-detected chlorella solution are divided into an experimental group and a blank control group; the experimental group is to add a certain amount of the apoptosis inducer to the to-be-detected alga solution, and further, when the apoptosis inducer is the 6a1, since the 6a1 contains a self culture medium, such as a 2216E culture medium containing autoclaving, in order to verify the influence of the 6a1 containing culture medium on the apoptosis of the alga, the experimental group is to add a certain amount of the 6a1 to the to-be-detected alga solution, and the blank control group is to add the 6a1 culture medium (i.e., the inhibitor contained in 6a1 is not added, and only the culture medium contained in 6a1 is added, such as the 2216E culture medium containing autoclaving) to the to-be-detected alga solution in the same amount as the experimental group.
Of course, the experimental group and the blank control group are cultured under the same conditions after being set.
In order to further improve the detection accuracy, 2-4 parallel samples are arranged in each group of the experimental group and the blank control group, and the content of the dinoflagellate cells in each part of the dinoflagellate solution to be detected is controlled to be the same so as to ensure the comparability of detection. In one embodiment, the content of dinoflagellate cells in each of the dinoflagellate solutions to be detected is 105-106cells/ml; in a specific embodiment, the dinoflagellate in the dinoflagellate solution to be detected may be, but is not limited to, Alexandrium tamarense. The content of the dinoflagellate cells in the dinoflagellate liquid to be detected and the number of the parallel samples are controlled, so that the detection accuracy is improved. In addition, the medium of the algal solution to be detected may be, but not limited to, an autoclaved f/2 medium.
In step S02, the centrifugation and suspension treatment of the chlorella solution subjected to the apoptosis-inducing treatment may be performed as follows:
will be passed throughAnd (3) carrying out centrifugal treatment on the dinoflagellate liquid subjected to the apoptosis induction treatment, collecting precipitated dinoflagellate cells, and then carrying out suspension treatment on the precipitated dinoflagellate cells. The solution used for suspension treatment may be, but is not limited to, deionized water to dilute the Binding Buffer (AnnexinV/FITC Binding Buffer) at 1: 3. In one embodiment, the concentration of the dinoflagellate cells in the dinoflagellate cell concentrate can be controlled to 105-106cells/ml, specifically 105cells/ml。
In the step S03, annexin v/FITC is added to the dinoflagellate cell concentrated solution to dye dinoflagellate cells, and in an embodiment, the annexin v/FITC is added to the dinoflagellate cell concentrated solution according to a ratio of 1 to 5 μ l of annexin v/FITC to 100 μ l of dinoflagellate cell concentrated solution, wherein a dinoflagellate cell concentration in the dinoflagellate cell concentrated solution is 105cells/ml. In addition, during the process of adding annexin V/FITC to realize the dyeing incubation treatment of the dinoflagellate cells, the incubation treatment can be carried out under the condition of keeping out of the light at room temperature, for example, for 5 min.
After annexin V/FITC dyes the dinoflagellate cell concentrated solution, adding propidium iodide into the dinoflagellate cell concentrated solution for dyeing, wherein in one embodiment, the propidium iodide is added into the dinoflagellate cell concentrated solution according to a ratio of 0.1-0.5 μ g of propidium iodide per 100 μ l of dinoflagellate cell concentrated solution, and the dinoflagellate cell concentrated solution has a dinoflagellate cell concentration of 105cells/ml. The control of the dyeing degree of the dinoflagellate cells is controlled by controlling the addition amount of the dye, so that the regulation and control of the dyeing effect of the dinoflagellate cells are controlled, and the accuracy of the final detection of the apoptosis rate of the dinoflagellate cells is improved.
In the step S04, the method for generating the volume/aspect ratio scattergram of the dinoflagellate cells comprises the steps of:
taking the stained dinoflagellate cell sap in the step S03, loading the stained dinoflagellate cell sap into the imaging flow cytometer, adjusting the laser power of the imaging flow cytometer to the upper power limit, adjusting the laser power to gradually reduce until fluorescence saturation is avoided, and then generating the volume/aspect ratio scatter diagram.
Wherein the step-down of the power of the laser controlling the imaging flow cytometer from the upper limit until fluorescence saturation is avoided is to obtain a clear high quality volume/Aspect Ratio scattergram (Area/Aspect Ratio scattergram). In one embodiment, the original maximum pixel (raw max pixel) of the volume/aspect ratio scatterplot generated is < 4096 by adjusting the laser power to decrease from an upper limit until the laser power is 4-20MV when fluorescence saturation is avoided. A clear high quality volume/aspect ratio scattergram was obtained by adjusting the laser power to 4-20 MV. The fluorescence saturation refers to that in general, a substance is excited to generate fluorescence, the intensity of the fluorescence increases with the increase of the excitation intensity, but when the excitation intensity is large to a certain degree, the intensity of the fluorescence starts to trend to a constant value and does not increase due to the increase of the excitation intensity, and the fluorescence saturation is called to occur.
On the basis of adjusting the laser power of the imaging flow cytometer, the sensitivity of the instrument to fluorescence of each channel can be increased by reducing the flow rate of the sample injection of the dinoflagellate staining cell sap in the imaging flow cytometer, for example, in a further embodiment, the flow rate of the sample injection of the dinoflagellate staining cell sap in the imaging flow cytometer is controlled to be 1 μ l/min to 4 μ l/min.
Therefore, in a specific embodiment, when the dinoflagellate in the dinoflagellate liquid to be detected is dinoflagellate, the dinoflagellate cell population having an aspect ratio of 0 to 0.6 is selected according to the volume/aspect ratio scattergram generated in step S04. When the dinoflagellates in the dinoflagellate liquid to be detected are other than the dinoflagellate liquid, all dinoflagellate cell populations in the volume/aspect ratio scattergram generated in step S04 may be selected.
In one embodiment, the method for generating the annexin v/PI scattergram according to the dinoflagellate cell population selected in step S04 comprises the following steps:
and (3) performing fluorescence compensation treatment on the dinoflagellate cell population by using analysis software of the imaging flow cytometer, and then generating the annexinV/PI cell scattergram.
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:
obtaining chlorophyll monofluorescence sample data to generate a monofluorescence sample data file, selecting a Compensation (Compensation) window in the analysis software, importing the chlorophyll monofluorescence sample data file, then automatically generating a Compensation Matrix (Compensation Matrix), adjusting the numerical value of the red marking Matrix (Matrix), completing the Compensation file after image verification, importing the Compensation file when analyzing data to realize fluorescence Compensation of the dinoflagellate cell population.
In a specific embodiment, in generating the annexin V/PI cell scattergram, annexin V fluorescence is the second channel, PI fluorescence is the fourth channel, and chlorophyll contained in dinoflagellate cells autofluorescence is the fifth channel. In the generated AnnexinV/PI cell scatter diagram, the abscissa of the plot is Intensity _ MC _ annexv, and the ordinate of the plot is Intensity _ MC _ PI. Therefore, the AnnexinV/PI cell scattergram can verify that the dinoflagellate cells are divided into four subgroups by images of the dinoflagellate cells, wherein the four subgroups are a normal living cell subgroup, an early apoptosis cell subgroup, an late apoptosis cell subgroup and a necrosis cell subgroup.
In addition, the imaging flow cytometer in the step S05 may be, but is not limited to
FlowSight, the analysis software may be analysis software idea, etc.
In the step S05, since the AnnexinV fluorescence and the PI fluorescence generated in the step S05 of the annexiv/PI scattergram and the chlorophyll autofluorescence contained in the dinoflagellate cells are respectively displayed in different fifth channels, the method for detecting dinoflagellate cell apoptosis can effectively distinguish the annexiv fluorescence and the propidium iodide fluorescent chlorophyll autofluorescence, thereby directly and quantitatively detecting the number of dinoflagellate cells undergoing apoptosis in the dinoflagellate cells to be detected, and obtaining the early apoptosis rate of the cells. In one embodiment, the calculating of the dinoflagellate apoptosis rate in the dinoflagellate solution to be detected according to the dinoflagellate apoptosis cell number is according to the following formula:
the dinoflagellate apoptosis rate ═ CX/C0×100％
In the formula CXThe number of apoptotic cells in the xth to-be-detected alga solution is counted; c0The total number of the dinoflagellate cells in the xth dinoflagellate solution to be detected is collected by the imaging flow cytometer.
Therefore, the method for detecting dinoflagellate apoptosis utilizes annexin V/FITC and PI dyes to dye dinoflagellate cells, and can effectively distinguish annexin V fluorescence and propidium iodide fluorescent chlorophyll autofluorescence through an imaging flow cytometer according to the characteristics and apoptosis characteristics of the dinoflagellate cells, so that the number of dinoflagellate cells to be detected undergoing apoptosis can be directly and quantitatively measured, and the early apoptosis rate of the cells can be obtained. The method effectively overcomes the problems that the existing annexin V/PI double staining method cannot effectively distinguish the spontaneous Chla red fluorescence of the dinoflagellate cells from the fluorescence emitted by PI and cannot visually observe the appearance characteristics of the dinoflagellate cells, so that partial dinoflagellate cell groups in different characteristic limits are difficult to distinguish and the fluorescence signals in single cells cannot be accurately positioned.
The present invention will now be described in further detail with reference to specific examples.
Example 1
This example provides a method for detecting dinoflagellate apoptosis. The method for detecting the dinoflagellate apoptosis comprises the following steps:
s11: taking 4 volumetric flasks, adding into the Alexandrium tamarense liquid (the culture medium of the Alexandrium tamarense liquid is 2216E culture medium), dividing the 4 volumetric flasks into 2 groups: experimental group (2 bottles), blank control group (2 bottles); the numbers of the dinoflagellate cells in the experimental group and the blank group are consistent; wherein 6A1 (6A1 contains 10) with 5 vol% is added into the volumetric flask of the experimental group8cells/ml suppressor cells, and the culture medium is 2216E culture medium); 5% by volume of 2216E medium 6A1 was added to the control volumetric flask (i.e. the control contained no 6A1 medium)Bacteriostatic cells); performing supernatant induction on the experimental group and the control group for 120min under the same condition;
s12: centrifuging each group of dinoflagellate cells treated in step S11 at 1000rpm for 5min, precipitating the cells, removing supernatant, adding about 1ml of PBS precooled at 4 ℃, resuspending the cells, centrifuging the precipitated cells again, and removing supernatant; diluting binding buffer (4ml 4 x binding buffer +12ml deionized water) with deionized water at a ratio of 1:3, and resuspending the cells with 1 x binding buffer to obtain the dinoflagellate cell concentrate;
s13: collecting 100 μ l of the dinoflagellate cell concentrate (dinoflagellate cell concentration is 2 × 10)5cells/ml), adding 5 mul of annexin V/FITC, uniformly mixing, incubating at room temperature in a dark place, and adding 10 mul of 20ug/ml propidium iodide solution (PI) after 5min to obtain dinoflagellate staining cell sap;
s14: immediately detecting on a machine after sampling, adjusting the power of a laser to be 4MV, and generating an Area/Aspect Ratio scatter diagram by the flow rate of the stained dinoflagellate cell sap injected in the imaging flow cytometer to be 3 mu l/min, and acquiring the Area of 500-2500 mu m3A population of cells with an Aspect Ratio between 0-0.6;
s15: by using
The analysis software idea of FlowSigt firstly carries out fluorescence compensation on the cell population in the step S13, and then generates an annexin V/PI cell scatter diagram, wherein the analysis and comparison in the diagram obtain the apoptosis region of the Alexandrium tamarense, and the result diagram is the annexin V/PI scatter diagram; wherein, the annexin V/PI scatter diagram of the control group is shown in figure 2, and the annexin V/PI scatter diagram of the experimental group is shown in figure 3;
s16: according to the calculation formula of the dinoflagellate apoptosis rate in fig. 2 and 3, the control group is 0% at 0min, the control group is 3.21% at 120min, the experimental group is 0% at 0min, and the experimental group is 47.59% at 120 min.
And necrotic cells are represented by AnnexinV-/PI + in the upper left corner region Q1 in FIGS. 2 and 3; the upper right corner region Q2 is annexin V +/PI +, indicating late apoptotic cells; the lower left corner region Q3 is annexin V-/PI-, indicating a normal living cell; the lower right region Q4 is annexin V +/PI-, indicating early apoptotic cells. Further, for the single dinoflagellate cell images in different states, there are four images per cell, namely BF (bright field), AnnexinV (pseudo-green), PI (pseudo-orange), Chla (pseudo-red), and BF/PI/annexiv, as shown in fig. 4.
Example 2
This example provides a method for detecting dinoflagellate apoptosis. The method for detecting the dinoflagellate apoptosis comprises the following steps:
s11: taking 4 volumetric flasks, adding into the Alexandrium tamarense liquid (the culture medium of the Alexandrium tamarense liquid is 2216E culture medium), dividing the 4 volumetric flasks into 2 groups: experimental group (2 bottles), control group (2 bottles); the numbers of the dinoflagellate cells in the experimental group and the blank group are consistent; wherein 6A1 (wherein 6A1 contains 10% by volume) is added into the volumetric flask of the experimental group in an amount of 10% by volume8cells/ml suppressor cells, and the culture medium is 2216E culture medium); adding 2216E culture medium of 6A1 with the volume of 10% into the volumetric flask of the control group (namely, the control group does not contain the bacteriostatic bacteria cells of 6A 1); performing supernatant induction on the experimental group and the control group under the same condition for 60 min;
s12: centrifuging each group of dinoflagellate cells treated in step S11 at 1000rpm for 5min, precipitating the cells, removing supernatant, adding about 1ml of PBS precooled at 4 ℃, resuspending the cells, centrifuging the precipitated cells again, and removing supernatant; diluting binding buffer (4ml 4 x binding buffer +12ml deionized water) with deionized water at a ratio of 1:3, and resuspending the cells with 1 x binding buffer to obtain the dinoflagellate cell concentrate;
s13: collecting 100 μ l of the dinoflagellate cell concentrate (dinoflagellate cell concentration is 2 × 10)5cells/ml), adding 1 μ l of annexin V/FITC, mixing uniformly, incubating at room temperature in dark, adding 20 μ l of 20ug/ml propidium iodide solution (PI) after 5min, and obtaining stained dinoflagellate cell sap;
s14: immediately detecting on a machine after sampling, adjusting the power of a laser to be 4MV, and generating Area/A by the flow velocity of the sample introduction of the dinoflagellate staining cell sap in the imaging flow cytometer to be 4 mul/minThe feature Ratio scatter diagram is obtained at 500-2500 μm for Area3A population of cells with an Aspect Ratio between 0-0.6;
s15: by using
The analysis software idea of FlowSigt firstly carries out fluorescence compensation on the cell population in the step S13, and then generates an annexin V/PI cell scatter diagram, wherein the analysis and comparison in the diagram obtain the apoptosis region of the Alexandrium tamarense, and the result diagram is the annexin V/PI scatter diagram; wherein, the annexin V/PI scatter diagram of the control group is shown in figure 5, and the annexin V/PI scatter diagram of the experimental group is shown in figure 6;
s16: according to the above calculation formula of the dinoflagellate apoptosis rate in fig. 2 and 3, the control group was 0% at 0min, 0.37% at 60min, 0% at 0min and 63.6% at 60 min.
In summary, the methods for detecting dinoflagellate apoptosis in the embodiments of the present invention can effectively distinguish AnnexinV fluorescence and propidium iodide fluorescent chlorophyll autofluorescence, so as to directly and quantitatively measure the number of dinoflagellate cells undergoing apoptosis in dinoflagellate cells to be detected, thereby obtaining the early apoptosis rate of the cells. Therefore, compared with the traditional detection method, the method is more convenient, quicker and more accurate.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (6)
1. A method for detecting dinoflagellate apoptosis is characterized by comprising the following steps:
carrying out apoptosis induction treatment on the dinoflagellate liquid to be detected by adopting an apoptosis inducer;
carrying out centrifugal suspension treatment on the dinoflagellate solution subjected to apoptosis induction treatment to obtain a dinoflagellate cell concentrated solution;
adding annexin V/FITC into the dinoflagellate cell concentrated solution, performing incubation treatment, and then continuously adding propidium iodide for dyeing treatment to obtain dyed dinoflagellate cell solution;
performing flow detection on the dinoflagellate cells in the stained dinoflagellate cell sap by using an imaging flow cytometer to generate a volume/aspect ratio scattergram of the dinoflagellate cells, and selecting a dinoflagellate cell population with an aspect ratio of 0-0.6 according to the volume/aspect ratio scattergram to obtain a required dinoflagellate cell population;
obtaining chlorophyll monofluorescence sample data to generate a monofluorescence sample data file, selecting a compensation window in analysis software of the imaging flow cytometer, importing the monofluorescence sample data file, then automatically generating a compensation matrix, adjusting a matrix numerical value marked with red, completing the compensation file after image verification, importing the compensation file when analyzing data to realize fluorescence compensation of the dinoflagellate cell population, and then generating an annexin V/PI cell scatter diagram; in the generation of the AnnexinV/PI cell scattergram, the AnnexinV fluorescence is a second channel, the PI fluorescence is a fourth channel, and the chlorophyll autofluorescence contained in dinoflagellate cells is a fifth channel;
and obtaining the number of the dinoflagellate apoptotic cells in the to-be-detected dinoflagellate liquid according to the annexinV/PI scatter diagram, and calculating the dinoflagellate apoptosis rate in the to-be-detected dinoflagellate liquid according to the number of the dinoflagellate apoptotic cells.
2. The method of claim 1, wherein: a method of generating the volume/aspect ratio scattergram of the dinoflagellate cells comprising the steps of:
and taking the stained dinoflagellate cell sap, loading the stained dinoflagellate cell sap into the imaging flow cytometer, adjusting the power of a laser of the imaging flow cytometer to the upper limit of the power, debugging the power of the laser to gradually reduce until the fluorescence saturation is avoided, and then generating the volume/aspect ratio scatter diagram.
3. The method of claim 2, wherein: debugging the power of the laser to be gradually reduced until the power of the laser is 4-20MV when the fluorescence saturation is avoided;
the flow rate of the sample injection of the dinoflagellate staining cell sap in the imaging flow cytometer is 1-4 mul/min.
4. A method according to any one of claims 1 to 3, wherein: calculating the dinoflagellate apoptosis rate in the to-be-detected dinoflagellate liquid according to the dinoflagellate apoptosis cell number according to the following formula:
the dinoflagellate apoptosis rate% = CX/C0×100%;
In the formula CXThe number of apoptotic cells in the xth to-be-detected alga solution is counted; c0The total number of the dinoflagellate cells in the xth dinoflagellate solution to be detected is collected by the imaging flow cytometer.
5. A method according to any one of claims 1-3, characterized in that: the dinoflagellate in the dinoflagellate liquid to be detected is Alexandrium tamarense.
6. A method according to any one of claims 1-3, characterized in that: the concentration of the dinoflagellate cells in the dinoflagellate cell concentrated solution is 105-106cells/ml; and/or
The annexin V/FITC is added into the dinoflagellate cell concentrated solution according to the proportion that 1-5 mul of annexin V/FITC is added into 100 mul of dinoflagellate cell concentrated solution, wherein the dinoflagellate cell concentration in the dinoflagellate cell concentrated solution is 105-106cells/ml; and/or
The propidium iodide is added into the dinoflagellate cell concentrated solution according to the proportion that 0.1-0.5 mu g of propidium iodide is added into 100 mu l of dinoflagellate cell concentrated solution, wherein the dinoflagellate cell concentration in the dinoflagellate cell concentrated solution is 105-106cells/ml。
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