CN110018143A - The method for detecting dinoflagellate Apoptosis - Google Patents
The method for detecting dinoflagellate Apoptosis Download PDFInfo
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Abstract
The invention discloses a kind of methods for detecting dinoflagellate Apoptosis.The method of the detection dinoflagellate Apoptosis including the following steps: apoptosis processing is carried out to dinoflagellate cell；Concentration is carried out to the dinoflagellate cell of apoptosis processing；AnnexinV, PI dyeing processing are carried out to dinoflagellate cell concentration liquid；Imaging flow cytometer detection processing is carried out to dyeing dinoflagellate cell liquid；The apoptosis rate of dinoflagellate cell is calculated according to AnnexinV/PI scatter plot.The method that the present invention detects dinoflagellate Apoptosis can effectively distinguish AnnexinV fluorescence, propidium iodide fluorescence, chlorophyll autofluorescence by the way that flow cytometer is imaged, to which direct quantitative measures the dinoflagellate number of cells of generation apoptosis in dinoflagellate cell to be measured, to obtain early apoptosis of cells rate, but also have quantitative reliable, the advantages that analysis is accurate, and as a result precision is high.
Description
Technical field
The invention belongs to Cell Measurement Technique fields, and in particular to a method of detection dinoflagellate Apoptosis.
Background technique
Red tide is a kind of harmful ecological phenomenon for causing water body to change color after planktonic organism is largely broken out, and can be caused red
The type that tide occurs has more than 300 kinds, such as red tide nocuousness dinoflagellate tower Ma Alexandria dinoflagellate (Alexandrium
Tamarense) it can discharge paralytic shellfish poison (paralyticshell fish poisoning, PSP), and shellfish poison enters ring
Border later constitutes a serious threat to marine economy and human health.Programmed death (Programmed cell death, PCD)
It is the autonomous orderly death of cell gene regulation when growing or by environment-stress, in order to effectively control red tide, understands red tide
Dinoflagellate Apoptosis has significant meaning.
Mainly there is cell membrane phosphinylidyne rouge serine (PS) detection using more method of cell apoptosis at present,
Caspase Enzyme assay, DNA marker detection method in situ, ROS detection.Wherein, with cell membrane phosphinylidyne rouge serine (PS) detection
For method, principle is to detect Apoptosis using the bis- dyes of AnnexinV/PI.Cell is when apoptosis early stage, cell
Phosphinylidyne rouge serine (PS) in film can be transferred to outside cell membrane out of cell membrane, it is made to be exposed to membrane surface, and
AnnexinV is Ca 2+-dependent-phospholipid-binding proteins, it can be specifically bound with PS, and PI is a kind of nucleic acid dye, it cannot
Through complete cell membrane, but to the cell and dead cell of apoptosis middle and advanced stage, PI can be such that nucleus incarnadines through cell membrane.
Therefore normal cell, apoptosis early stage, apoptosis advanced stage and non-viable non-apoptotic cell can be distinguished using the method for the bis- dyes of AnnexinV/PI.
Flow cytometer is mainly used in medical research such as cell viability, cellular identification.Apoptosis and cell signal
Transduction etc. is widely used using the method for the bis- dye detection Apoptosis PS of AnnexinV/PI.But this method is applied to inspection
There is limitation when surveying dinoflagellate class Apoptosis, because dinoflagellate cell has autofluorescence, ordinary stream cell instrument is universal
Cannot distinguish between the spontaneous chlorophyll of dinoflagellate cell (Chla) red fluorescence, (PI molecular formula is C with propidium iodide27H34I2N4) issue
Fluorescence, and the external appearance characteristic of dinoflagellate cell also can not be intuitively observed while using, when analyzing sample data
It is difficult to differentiate between the part dinoflagellate cell colony in different characteristic boundary, it also can not fluorescence letter in precise positioning individual cells
Number, it is not high so as to cause result precision, but also have the defects that technical requirements are high, sample consumption is big.
Summary of the invention
It is an object of the invention to overcome the deficiency of the prior art, a kind of side for detecting dinoflagellate Apoptosis is provided
Method cannot distinguish between the fluorescence that the spontaneous Chla red fluorescence of dinoflagellate cell and PI are issued to solve existing ordinary stream cell instrument, from
And the technical problem for causing the accuracy of Apoptosis testing result not high.
In order to realize the goal of the invention, the present invention provides a kind of methods for detecting dinoflagellate Apoptosis.The detection
The method of dinoflagellate Apoptosis includes the following steps:
Dinoflagellate liquid to be detected is subjected to apoptosis-inducing processing using cell death inducer；
Centrifugation suspension processing, dinoflagellate cell concentration are carried out to the dinoflagellate liquid for handling processing through the apoptosis-inducing
Liquid；
After adding AnnexinV/FITC into the dinoflagellate cell concentration liquid and carrying out incubation processing, it is further continued for that iodine is added
Change the third pyridine and carry out dyeing processing, obtains dyeing dinoflagellate cell liquid；
The dinoflagellate cell characteristic in the dyeing dinoflagellate cell liquid will be analyzed using imaging flow cytometer, is generated
AnnexinV/PI scatter plot, according to the scatter plot choose cell colony, then according to the cell colony of selection obtain by
The dinoflagellate Apoptosis number in dinoflagellate sample is surveyed, dinoflagellate cell is calculated according to the dinoflagellate Apoptosis number
Apoptosis rate.
Compared with prior art, the method that the present invention detects dinoflagellate Apoptosis is contaminated using phosphatidyl serine dyestuff and PI
Material dyes dinoflagellate cell, according to dinoflagellate cell self-characteristic and Apoptosis feature, by the way that flow cytometer energy is imaged
It is enough effectively to distinguish AnnexinV fluorescence, propidium iodide fluorescence chlorophyll autofluorescence, so as to measure dinoflagellate to be measured thin for direct quantitative
The dinoflagellate number of cells of apoptosis occurs in born of the same parents, to obtain early apoptosis of cells rate, but also with quantitatively reliably, analysis is quasi-
Really, the advantages that as a result precision is high.
Detailed description of the invention
Fig. 1 is the method and process flow diagram that the embodiment of the present invention detects dinoflagellate Apoptosis；
Fig. 2 be the embodiment of the present invention 1 in control group dinoflagellate cell different times AnnexinV/PI scatter plot；Wherein,
Fig. 2A is AnnexinV/PI scatter plot of the control group dinoflagellate cell in 0min, and Fig. 2 B is control group dinoflagellate cell in 120min
When AnnexinV/PI scatter plot；
Fig. 3 be the embodiment of the present invention 1 in experimental group dinoflagellate cell different times AnnexinV/PI scatter plot；Wherein,
Fig. 3 A is AnnexinV/PI scatter plot of the experimental group dinoflagellate cell in 0min, and Fig. 3 B is experimental group dinoflagellate cell in 120min
When AnnexinV/PI scatter plot；
Fig. 4 is the image of single dinoflagellate cell in AnnexinV/PI scatter plot in the embodiment of the present invention 1；
Fig. 5 be the embodiment of the present invention 2 in control group dinoflagellate cell different times AnnexinV/PI scatter plot；Wherein,
Fig. 5 A is AnnexinV/PI scatter plot of the control group dinoflagellate cell in 0min, and Fig. 5 B is control group dinoflagellate cell in 60min
When AnnexinV/PI scatter plot；
Fig. 6 be the embodiment of the present invention 2 in experimental group dinoflagellate cell different times AnnexinV/PI scatter plot；Wherein,
Fig. 6 A is AnnexinV/PI scatter plot of the experimental group dinoflagellate cell in 0min, and Fig. 6 B is experimental group dinoflagellate cell in 60min
When AnnexinV/PI scatter plot.
Specific embodiment
In order to which technical problems, technical solutions and advantageous effects to be solved by the present invention are more clearly understood, below in conjunction with
Embodiment, the present invention will be described in further detail.It should be appreciated that specific embodiment described herein is only used to explain
The present invention is not intended to limit the present invention.
In the embodiment of the present invention, hereafter noun is made as described below.
Phosphatide phthalein serine: its inside for being normally at cell membrane (Phosphatidlserinea, PS), but in cell tune
The early stage died, PS can be exposed in extracellular environment from the surface of cell membrane turned inside out to cell membrane.
Annexin V: being a kind of Ca that molecular weight is 35-36kD2+Dependence cardiolipin binding protein, can be with PS high-affinity
Specific binding.Annexin V is subjected to fluorescein (FITC) label, it is sharp using the Annexin V that is marked as fluorescence probe
It can detect the generation of natural death of cerebral cells with flow cytometer or fluorescence microscope.
Propidium iodide (propidine iodide, PI): being a kind of nucleic acid dye, it cannot penetrate complete cell membrane,
But in the cell and dead cell of apoptosis middle and advanced stage, PI can make the red dye of nucleus through cell membrane.Therefore by Annexin V with
PI matching uses, so that it may distinguish the cell in apoptosis early advanced stage and dead cell.
The embodiment of the invention provides a kind of methods for detecting dinoflagellate Apoptosis.The side of the detection dinoflagellate Apoptosis
Method includes the following steps:
S01. apoptosis processing is carried out to dinoflagellate cell: dinoflagellate liquid to be detected is subjected to cell using cell death inducer and is withered
Die induction processing；
S02. centrifugation suspension processing: the institute that will be handled through the apoptosis-inducing is carried out to the dinoflagellate cell of apoptosis processing
It states dinoflagellate liquid and carries out centrifugation suspension processing, obtain dinoflagellate cell concentration liquid；
S03. AnnexinV, PI dyeing processing are carried out to dinoflagellate cell concentration liquid: added in Xiang Suoshu dinoflagellate cell concentration liquid
After adding AnnexinV/FITC and carrying out incubation processing, it is further continued for addition propidium iodide and carries out dyeing processing, it is thin to obtain dyeing dinoflagellate
Cytosol；
S04. imaging flow cytometer detection processing is carried out to dyeing dinoflagellate cell liquid: using imaging flow cytometer to the dyeing
Dinoflagellate cell in dinoflagellate cell liquid carries out flow cytometer detection, needed for generating volume/aspect ratio scatter plot of dinoflagellate cell to obtain
Dinoflagellate cell colony；AnnexinV/PI scatter plot is generated further according to the dinoflagellate cell colony of selection；
S05. the apoptosis rate of dinoflagellate cell is calculated according to AnnexinV/PI scatter plot: being dissipated according to the AnnexinV/PI
Point diagram obtains the dinoflagellate Apoptosis number in the dinoflagellate liquid to be detected, according to the dinoflagellate Apoptosis number
Calculate the dinoflagellate apoptosis rate in the dinoflagellate liquid to be detected.
Wherein, described after the cell death inducer is added in the dinoflagellate liquid to be detected in the step S01
Cell death inducer can have an effect to induce the dinoflagellate apoptosis in dinoflagellate liquid to be detected.Wherein, described thin
Born of the same parents' inducer of apoptosis, which can according to need, selects different cell death inducers.As in an embodiment, the Apoptosis is lured
Leading agent may include molten dinoflagellate bacterium, H2O2, at least one of virus.Wherein, the molten dinoflagellate bacterium (6A1) is by this seminar
It is located away from the red tide seawater that Tai Pang Wan Dongshan, Shenzhen sea area is broken out.Certainly, molten dinoflagellate bacterium can also be other algae-lysing bacteriums.
Certainly it can realize that the apoptosis of the dinoflagellate cell, such as building nitrogen hunger, iron starvation are realized with the environment of component Apoptosis
The apoptosis of the dinoflagellate cell.
In addition, the type for the cell death inducer chosen is different, it is added into the dinoflagellate liquid to be detected
Amount is different, but the amount into the dinoflagellate liquid to be detected is added no more than described to be detected in the general cell death inducer
The 10% of dinoflagellate liquid volume ratio.As in one embodiment, the cell death inducer is added in the dinoflagellate liquid to be detected
Volume ratio be 1%-10%.
For the accuracy of detection, in an embodiment, the dinoflagellate liquid to be detected is divided into several pieces, by the institute of several pieces
Dinoflagellate liquid to be detected is stated to be divided into including experimental group and blank control group；Wherein, the experimental group is to the dinoflagellate liquid to be detected
Middle a certain amount of cell death inducer of addition, further, when the cell death inducer selects the 6A1,
Since 6A1 contains the culture medium of itself, such as contain autoclaved 2216E culture medium, in order to verify the 6A1 containing culture medium pair
The influence of dinoflagellate Apoptosis, therefore, the experimental group are to add a certain amount of 6A1 into the dinoflagellate liquid to be detected,
The blank control control group be added into the dinoflagellate liquid to be detected with the 6A1 culture medium of experimental group isodose (
It is not add inhibition bacterium contained by 6A1, only adds culture medium contained by 6A1, such as autoclaved 2216E culture medium).
Setting completed is placed under the same terms and carries out culture processing for certain experimental group and blank control group.
In order to further increase the accuracy of detection, the experimental group is parallel with every group of setting 2-4 of blank control group
Sample, and the dinoflagellate cell content control in every portion dinoflagellate liquid to be detected is identical, to guarantee the comparability of detection.One
In embodiment, the dinoflagellate cell content in every portion dinoflagellate liquid to be detected is 105-106cells/ml；In specific embodiment
In, dinoflagellate in the dinoflagellate liquid to be detected can be with but not just for Alexandrium tamarense.By controlling dinoflagellate liquid to be detected
In dinoflagellate cell content and Duplicate Samples quantity, with improve detection accuracy.In addition, the culture of the dinoflagellate liquid to be detected
Base can be with but not just for autoclaved f/2 culture medium.
In the step S02, centrifugation suspension processing is carried out to by the dinoflagellate liquid handled through the apoptosis-inducing
It can carry out as follows:
The dinoflagellate liquid handled through the apoptosis-inducing is subjected to centrifugal treating, collects the dinoflagellate cell of precipitating,
Then suspension processing is carried out to the dinoflagellate cell of precipitating.For suspend processing solution can with but not only deionized water by 1:3
It dilutes combination buffer (AnnexinV/FITC Binding Buffer).In one embodiment, the dinoflagellate cell concentration is obtained
The concentration of dinoflagellate cell in liquid can control 105-106Cells/ml specifically can be 105cells/ml。
In the step S03, in Xiang Suoshu dinoflagellate cell concentration liquid add AnnexinV/FITC realize to dinoflagellate cell into
Row dyeing is handled, and in an embodiment, the AnnexinV/FITC is that 1- is added according to dinoflagellate cell concentration liquid described in every 100 μ l
The ratio of AnnexinV/FITC described in 5 μ l is added in the dinoflagellate cell concentration liquid, wherein the dinoflagellate cell concentration liquid
In dinoflagellate cell concentration be 105cells/ml.Dinoflagellate cell is contaminated in addition, addition AnnexinV/FITC realization is added
Color is incubated in treatment process, and the incubation processing can carry out under the conditions of room temperature is protected from light, and is such as incubated for 5min.
After AnnexinV/FITC carries out the dinoflagellate cell concentration liquid to dye and be disposed, then to the dinoflagellate
Propidium iodide is added in cell concentration liquid and carries out dyeing processing, and in an embodiment, the propidium iodide is according to described in every 100 μ l
The ratio that propidium iodide described in 0.1-0.5 μ g is added in dinoflagellate cell concentration liquid is added in the dinoflagellate cell concentration liquid, wherein
Dinoflagellate cell concentration in the dinoflagellate cell concentration liquid is 105cells/ml.Additive amount by controlling dyestuff is controlled to first
The control of frustule dye levels, so that the regulation of dinoflagellate cell dyeing effect is controlled, to improve final dinoflagellate Apoptosis
The accuracy of rate detection.
In the step S04, the volume/aspect ratio scatter plot method for generating the dinoflagellate cell includes following step
It is rapid:
It takes and flow cytometer is imaged described in dyeing dinoflagellate cell liquid described in step S03 and upper machine, then the imaging is flowed
The laser power of formula cell instrument is adjusted to the upper limit of the power, then debugs the laser power and gradually reduces until avoiding fluorescence full
With then generate the volume/aspect ratio scatter plot.
Wherein, the power for controlling the laser of the imaging flow cytometer is gradually reduced by the upper limit until fluorescence is avoided to satisfy
Be volume/aspect ratio scatter plot (Area/Aspect Ratio scatter plot) to obtain clear high quality.One embodiment
In, the adjusting gradually reduced by the power to laser by the upper limit is until laser power when fluorescence being avoided to be saturated is
4-20MV, at this point, the original maximum pixel of the volume/aspect ratio scatter plot (the raw max pixel) < 4096 generated.It is logical
Overregulate volume/aspect ratio scatter plot that the laser power obtains clear high quality to 4-20MV.Wherein, fluorescence saturation is
Refer to that under normal circumstances, by excitation fluorescence will be occurred for substance, and the intensity of fluorescence can increase with the increase of excitation intensity, but
When excitation intensity greatly to a certain extent when, the intensity of fluorescence just starts to be intended to a steady state value, and no longer because of the increasing of excitation intensity
Increase greatly, at this moment we, which weigh up, has showed fluorescence saturation.
It, can also be by reducing the dye on the basis of adjusting the laser power of the imaging flow cytometer
Color dinoflagellate cell liquid flow velocity of sample introduction in the imaging flow cytometer realizes the sensitivity for increasing instrument to each channel fluorescence,
In further embodiment, controlling dyeing dinoflagellate cell liquid flow velocity of sample introduction in the imaging flow cytometer is 1 μ l/
min-4μl/min。
Therefore, in one embodiment, when the dinoflagellate in the dinoflagellate liquid to be detected is first dinoflagellate, and according to step
The volume generated in S04/aspect ratio scatter plot chooses aspect ratio in the dinoflagellate cell colony of 0-0.6.When described to be detected
When dinoflagellate in dinoflagellate liquid is other, it can choose according to institute in the volume/aspect ratio scatter plot generated in step S04
Some dinoflagellate cell colonys.
In one embodiment, the AnnexinV/PI is generated according to the dinoflagellate cell colony chosen in step S04 and is dissipated
The method of point diagram includes the following steps:
After doing fluorescence compensation deals to the dinoflagellate cell colony using the analysis software of the imaging flow cytometer, then
Generate the AnnexinV/PI cell scatter plot.
Wherein, the fluorescence is done to the dinoflagellate cell colony using the analysis software of the imaging flow cytometer to compensate
Method include the following steps:
It obtains chlorophyll list fluorescent sample data and generates single fluorescent sample data file, select to mend in the analysis software
It repays after (Compensation) window imports single fluorescent sample data file and automatically generates compensation matrix (Compensation
Matrix), red matrix (Matrix) numerical value is marked in adjustment, by completing compensation file after image authentication, in analysis data
When import the compensation file realization fluorescence compensation done to the dinoflagellate cell colony.
In a particular embodiment, in generating the AnnexinV/PI cell scatter plot, AnnexinV fluorescence is second logical
Road, PI fluorescence are fourth lane, and chlorophyll autofluorescence contained by dinoflagellate cell is in Five-channel.Described in generation
In AnnexinV/PI cell scatter plot, abscissa be Intensity_MC_AnnexinV, ordinate Intensity_
MC_PI.Therefore, the AnnexinV/PI cell scatter plot can be verified by the image to its dinoflagellate cell by its circle door
It is divided into four kinds of subgroups, respectively normal live cells subgroup, viable apoptotic cell subgroup, non-viable apoptotic cell subgroup, non-viable non-apoptotic cell
Subgroup.
In addition, the imaging flow cytometer in the step S05 can with but not just for
FlowSight, the analysis software can be analysis software ideas etc..
In the step S05, the AnnexinV/PI scatter plot AnnexinV fluorescence, the PI that are generated due to step S05
Chlorophyll autofluorescence contained by fluorescence and dinoflagellate cell is to show in different Five-channels respectively, therefore, this detection dinoflagellate
The method of Apoptosis can effectively distinguish AnnexinV fluorescence, propidium iodide fluorescence chlorophyll autofluorescence, thus directly fixed
The dinoflagellate number of cells that apoptosis occurs in dinoflagellate cell to be measured is measured, to obtain early apoptosis of cells rate.In one embodiment,
Calculating the dinoflagellate apoptosis rate in the dinoflagellate liquid to be detected according to the dinoflagellate Apoptosis number is according to such as
Lower formula calculates:
The dinoflagellate apoptosis rate %=CX/C0× 100%
C in formulaXFor the apoptotic cell number in xth part dinoflagellate liquid to be detected；C0For collected by imaging flow cytometer
Total dinoflagellate number of cells in xth part dinoflagellate liquid to be detected.
Therefore, the method for detection dinoflagellate Apoptosis described above is thin to dinoflagellate using AnnexinV/FITC and PI dyestuff
Born of the same parents dye, and according to dinoflagellate cell self-characteristic and Apoptosis feature, can effectively be distinguished by the way that flow cytometer is imaged
AnnexinV fluorescence, propidium iodide fluorescence chlorophyll autofluorescence, wither in dinoflagellate cell to be measured so that direct quantitative is measured
The dinoflagellate number of cells died, to obtain early apoptosis of cells rate, but also with quantitatively reliably, analysis is accurate, as a result precision
The advantages that high.Effectively overcome the method for the bis- dyes of existing AnnexinV/PI that can not effectively distinguish the spontaneous Chla of dinoflagellate cell red
Fluorescence that fluorescence and PI are issued and the external appearance characteristic that can not intuitively observe dinoflagellate cell, caused by be difficult to differentiate between in different
The part dinoflagellate cell colony of feature boundary and can not fluorescence signal in precise positioning individual cells problem.
Now in conjunction with specific example, the present invention will be described in further detail.
Embodiment 1
Present embodiments provide a kind of method for detecting dinoflagellate Apoptosis.The method packet of the detection dinoflagellate Apoptosis
Include following steps:
S11: 4 volumetric flasks are taken to be separately added into the tower Ma Alexandria dinoflagellate liquid (culture medium of tower Ma Alexandria dinoflagellate liquid
For 2216E culture medium), 4 volumetric flasks are divided into 2 groups: experimental group (2 bottles), blank control group (2 bottles)；And make to test
It is group, consistent to the dinoflagellate number of cells of blank photo group；Wherein, 6A1 (its of volume 5% is added in the volumetric flask of the experimental group
In, 6A1 contains 108Cells/ml inhibits bacterium cell, and culture medium is 2216E culture medium)；It is added in the volumetric flask of the control group
The 2216E culture medium (that is to say and be free of the inhibition bacterium cell of 6A1 in control group) of the 6A1 of volume 5%；By experimental group and control
Group carries out supernatant under the same conditions and induces 120min；
S12: by treated in step S11, each group dinoflagellate cell 1000rpm is centrifuged 5min, and sedimentation cell absorbs supernatant,
About 1ml, 4 DEG C of PBS being pre-chilled are added, cell is resuspended, again centrifugation cell, absorbs supernatant；It is dilute by 1:3 with deionized water
Combination buffer (4ml 4* combination buffer+12ml deionized water) is released, with 1* combination buffer again suspension cell, obtains institute
State dinoflagellate cell concentration liquid；
S13: take 100 μ l the dinoflagellate cell concentration liquid (dinoflagellate cell concentration be 2 × 105Cells/ml), 5 μ l are added
AnnexinV/FITC mix after in room temperature be protected from light incubation, the ofpropidium iodide solution of the 20ug/ml of 10 μ l is added after 5min
(PI), dyeing dinoflagellate cell liquid is obtained；
S14: upper machine testing immediately after sampling, adjustment laser power are 4MV, the dyeing dinoflagellate cell liquid it is described at
As sample introduction in flow cytometer flow velocity be 3 μ l/min, generate Area/Aspect Ratio scatter plot, obtain Area in 500-
2500μm3, cell colony of the Aspect Ratio between 0-0.6；
S15: it utilizesThe analysis software ideas of FlowSight first does the cell colony in step S13 glimmering
Light compensation, secondly regenerates AnnexinV/PI cell scatter plot, and analysis, which is compared, in figure show that tower Ma Alexandria dinoflagellate cell withers
The region died, result figure are AnnexinV/PI scatter plot；Wherein, the AnnexinV/PI scatter plot of control group is as shown in Fig. 2, reality
The AnnexinV/PI scatter plot for testing group is as shown in Figure 3；
S16:, according to the calculation formula of the dinoflagellate apoptosis rate, control can be calculated according to from Fig. 2 and Fig. 3
Group in 0min early tune rate be 0%, rate of early withering in 120min be 3.21%, experimental group early wither in 0min rate be 0%,
Rate of early withering when 120min is 47.59%.
And by upper left corner area Q1 in Fig. 2 and Fig. 3 be AnnexinV-/PI+, indicate non-viable non-apoptotic cell；Upper right comer region Q2
For AnnexinV+/PI+, non-viable apoptotic cell is indicated；Lower left corner region Q3 is AnnexinV-/PI-, indicates normal live cells；
Lower right field Q4 is AnnexinV+/PI-, indicates viable apoptotic cell.Further to the single dinoflagellate cell under different conditions
Image, for each cell there are four image, respectively BF (light field), AnnexinV (false green), PI (false orange), Chla are (false red
Color) and BF/PI/AnnexinV, it is specific as shown in Figure 4.
Embodiment 2
Present embodiments provide a kind of method for detecting dinoflagellate Apoptosis.The method packet of the detection dinoflagellate Apoptosis
Include following steps:
S11: 4 volumetric flasks are taken to be separately added into the tower Ma Alexandria dinoflagellate liquid (culture medium of tower Ma Alexandria dinoflagellate liquid
For 2216E culture medium), 4 volumetric flasks are divided into 2 groups: experimental group (2 bottles), control group (2 bottles)；And make experimental group, right
The dinoflagellate number of cells of blank photo group is consistent；Wherein, the 6A1 of volume 10% is added (wherein, in the volumetric flask of the experimental group
6A1 contains 108Cells/ml inhibits bacterium cell, and culture medium is 2216E culture medium)；Body is added in the volumetric flask of the control group
The 2216E culture medium (that is to say and be free of the inhibition bacterium cell of 6A1 in control group) of the 6A1 of product 10%；By experimental group and control group
Supernatant is carried out under the same conditions induces 60min；
S12: by treated in step S11, each group dinoflagellate cell 1000rpm is centrifuged 5min, and sedimentation cell absorbs supernatant,
About 1ml, 4 DEG C of PBS being pre-chilled are added, cell is resuspended, again centrifugation cell, absorbs supernatant；It is dilute by 1:3 with deionized water
Combination buffer (4ml 4* combination buffer+12ml deionized water) is released, with 1* combination buffer again suspension cell, obtains institute
State dinoflagellate cell concentration liquid；
S13: take 100 μ l the dinoflagellate cell concentration liquid (dinoflagellate cell concentration be 2 × 105Cells/ml), 1 μ l is added
AnnexinV/FITC mix after in room temperature be protected from light incubation, the ofpropidium iodide solution of the 20ug/ml of 20 μ l is added after 5min
(PI), dyeing dinoflagellate cell liquid is obtained；
S14: upper machine testing immediately after sampling, adjustment laser power are 4MV, the dyeing dinoflagellate cell liquid it is described at
As sample introduction in flow cytometer flow velocity be 4 μ l/min, generate Area/Aspect Ratio scatter plot, obtain Area in 500-
2500μm3, cell colony of the Aspect Ratio between 0-0.6；
S15: it utilizesThe analysis software ideas of FlowSight first does the cell colony in step S13 glimmering
Light compensation, secondly regenerates AnnexinV/PI cell scatter plot, and analysis, which is compared, in figure show that tower Ma Alexandria dinoflagellate cell withers
The region died, result figure are AnnexinV/PI scatter plot；Wherein, the AnnexinV/PI scatter plot of control group is as shown in figure 5, reality
The AnnexinV/PI scatter plot for testing group is as shown in Figure 6；
S16:, according to the calculation formula of above-mentioned dinoflagellate apoptosis rate, control can be calculated according to from Fig. 2 and Fig. 3
Group in 0min early tune rate be 0%, rate of early withering in 60min be 0.37%, experimental group early wither in 0min rate be 0%,
Rate of early withering when 60min is 63.6%.
The method of dinoflagellate Apoptosis is to sum up detected in each embodiment it is found that detection dinoflagellate provided in an embodiment of the present invention is thin
The method of born of the same parents' apoptosis can effectively distinguish AnnexinV fluorescence, propidium iodide fluorescence chlorophyll autofluorescence, thus direct quantitative
The dinoflagellate number of cells that apoptosis occurs in dinoflagellate cell to be measured is measured, to obtain early apoptosis of cells rate, but also has and determines
The advantages that amount is reliable, and analysis is accurate, and as a result precision is high.Therefore, more more convenient than traditional detection method accurate.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (10)
1. a kind of method for detecting dinoflagellate Apoptosis, which comprises the steps of:
Dinoflagellate liquid to be detected is subjected to apoptosis-inducing processing using cell death inducer；
The dinoflagellate liquid handled through the apoptosis-inducing is subjected to centrifugation suspension processing, obtains dinoflagellate cell concentration liquid；
After adding AnnexinV/FITC into the dinoflagellate cell concentration liquid and carrying out incubation processing, it is further continued for that iodate third is added
Pyridine carries out dyeing processing, obtains dyeing dinoflagellate cell liquid；
Flow cytometer detection is carried out to the dinoflagellate cell in the dyeing dinoflagellate cell liquid using imaging flow cytometer, it is thin to generate dinoflagellate
The volume of born of the same parents/aspect ratio scatter plot is to obtain required dinoflagellate cell colony；It is raw further according to the dinoflagellate cell colony of selection
At AnnexinV/PI scatter plot；
The dinoflagellate Apoptosis number in the dinoflagellate liquid to be detected is obtained according to the AnnexinV/PI scatter plot, according to
The dinoflagellate apoptosis rate in the dinoflagellate liquid to be detected is calculated according to the dinoflagellate Apoptosis number.
2. according to the method described in claim 1, it is characterized by: the volume/aspect ratio for generating the dinoflagellate cell dissipates
The method of point diagram includes the following steps:
Take and flow cytometer be imaged described in the dyeing dinoflagellate cell liquid and upper machine, then by it is described imaging flow cytometer laser
Device power is adjusted to the upper limit of the power, then debugs the laser power and gradually reduces up to avoiding fluorescence from being saturated, then generates institute
State volume/aspect ratio scatter plot.
3. according to the method described in claim 2, gradually reducing it is characterized by: debugging the laser power until avoiding glimmering
Laser power when light is saturated is 4-20 MV；
Dyeing dinoflagellate cell liquid flow velocity of sample introduction in the imaging flow cytometer is 1 μ l/min-4 μ l/min.
4. according to the method in claim 2 or 3, it is characterised in that: chosen in length and breadth according to the volume/aspect ratio scatter plot
Than the dinoflagellate cell colony in 0-0.6.
5. according to the method described in claim 1, it is characterized by: according to the generation of the dinoflagellate cell colony of selection
The method of AnnexinV/PI scatter plot includes the following steps:
After doing fluorescence compensation deals to the dinoflagellate cell colony using the analysis software of the imaging flow cytometer, regeneration
The AnnexinV/PI cell scatter plot.
6. the method according to claim 5, it is characterised in that: utilize the analysis software pair of the imaging flow cytometer
The method that the dinoflagellate cell colony does the fluorescence compensation includes the following steps:
It obtains chlorophyll list fluorescent sample data and generates single fluorescent sample data file, the selection compensation window in the analysis software
Mouth automatically generates compensation matrix after importing single fluorescent sample data file, and adjustment is marked red matrix numerical value, tested by image
Compensation file is completed after card, imported when analyze data the compensation file realize the dinoflagellate cell colony is done it is glimmering
Light compensation.
7. according to method described in claim 5 or 6, it is characterised in that: generating the AnnexinV/PI cell scatter plot
In, AnnexinV fluorescence is second channel, and PI fluorescence is fourth lane, and chlorophyll autofluorescence contained by dinoflagellate cell is logical the 5th
Road.
8. -3, any method of 5-6 according to claim 1, it is characterised in that: according to the dinoflagellate Apoptosis
Number, which calculates the dinoflagellate apoptosis rate in the dinoflagellate liquid to be detected, to be calculated according to following formula:
The dinoflagellate apoptosis rate %=CX/C0× 100%；
C in formulaXFor the apoptotic cell number in xth part dinoflagellate liquid to be detected；C0For xth collected by imaging flow cytometer
Total dinoflagellate number of cells in part dinoflagellate liquid to be detected.
9. -3, the described in any item methods of 5-6 according to claim 1, it is characterised in that: the dinoflagellate in the dinoflagellate liquid to be detected
For Alexandrium tamarense.
10. -3, the described in any item methods of 5-6 according to claim 1, it is characterised in that: in the dinoflagellate cell concentration liquid
Dinoflagellate cell concentration is 105-106cells/ml；And/or
The AnnexinV/FITC is that AnnexinV/ described in 1-5 μ l is added according to dinoflagellate cell concentration liquid described in every 100 μ l
The ratio of FITC is added in the dinoflagellate cell concentration liquid, wherein the dinoflagellate cell concentration in the dinoflagellate cell concentration liquid
It is 105-106cells/ml；And/or
The propidium iodide is the ratio that propidium iodide described in 0.1-0.5 μ g is added according to dinoflagellate cell concentration liquid described in every 100 μ l
Example is added in the dinoflagellate cell concentration liquid, wherein the dinoflagellate cell concentration in the dinoflagellate cell concentration liquid is 105-
106cells/ml。
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110542675A (en) * | 2019-09-30 | 2019-12-06 | 广州市锐博生物科技有限公司 | Apoptosis detection method and apoptosis detection kit |
| CN112322694A (en) * | 2020-10-21 | 2021-02-05 | 桂林理工大学 | Method for detecting green algae cell apoptosis |
| CN114295596A (en) * | 2021-12-30 | 2022-04-08 | 无锡代际生物科技有限公司 | Sperm quality multi-parameter detection kit |
| CN116183470A (en) * | 2022-12-22 | 2023-05-30 | 中国林业科学研究院林业研究所 | Woody plant cell programmed death detection method based on flow sorting |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101684161A (en) * | 2001-08-10 | 2010-03-31 | 阿赫姆生物系统公司 | System for detecting protease |
| US20130274442A1 (en) * | 2006-12-14 | 2013-10-17 | Georgia State University Research Foundation, Inc. | Analyte Sensors, Methods for Preparing and Using Such Sensors, and Methods of Detecting Analyte Activity |
| CN103712967A (en) * | 2013-12-27 | 2014-04-09 | 首都医科大学附属北京友谊医院 | Method for detecting activity of NK (Natural Killer) cell |
| CN105445248A (en) * | 2015-12-14 | 2016-03-30 | 上海应用技术学院 | Method for rapid determination of cyanobacteria cell apoptosis rate |
| CN108220357A (en) * | 2017-12-12 | 2018-06-29 | 深圳大学 | A kind of application of pyrroles -2- carboxylic acids and preparation method |
-
2019
- 2019-03-29 CN CN201910246905.XA patent/CN110018143B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101684161A (en) * | 2001-08-10 | 2010-03-31 | 阿赫姆生物系统公司 | System for detecting protease |
| US20130274442A1 (en) * | 2006-12-14 | 2013-10-17 | Georgia State University Research Foundation, Inc. | Analyte Sensors, Methods for Preparing and Using Such Sensors, and Methods of Detecting Analyte Activity |
| CN103712967A (en) * | 2013-12-27 | 2014-04-09 | 首都医科大学附属北京友谊医院 | Method for detecting activity of NK (Natural Killer) cell |
| CN105445248A (en) * | 2015-12-14 | 2016-03-30 | 上海应用技术学院 | Method for rapid determination of cyanobacteria cell apoptosis rate |
| CN108220357A (en) * | 2017-12-12 | 2018-06-29 | 深圳大学 | A kind of application of pyrroles -2- carboxylic acids and preparation method |
Non-Patent Citations (2)
| Title |
|---|
| TIAN X.-Q.等: "Globular Adiponectin Inhibits the Apoptosis of Mesenchymal Stem Cells Induced by Hypoxia and Serum Deprivation via the AdipoR1-Mediated Pathway", 《CELLULAR PHYSIOLOGY AND BIOCHEMISTRY》 * |
| VERONIKA DASHKOVA等: "Imaging flow cytometry for phytoplankton analysis", 《METHODS》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110542675A (en) * | 2019-09-30 | 2019-12-06 | 广州市锐博生物科技有限公司 | Apoptosis detection method and apoptosis detection kit |
| CN110542675B (en) * | 2019-09-30 | 2021-12-14 | 广州市锐博生物科技有限公司 | Apoptosis detection method and apoptosis detection kit |
| CN112322694A (en) * | 2020-10-21 | 2021-02-05 | 桂林理工大学 | Method for detecting green algae cell apoptosis |
| CN114295596A (en) * | 2021-12-30 | 2022-04-08 | 无锡代际生物科技有限公司 | Sperm quality multi-parameter detection kit |
| CN114295596B (en) * | 2021-12-30 | 2024-04-05 | 无锡代际生物科技有限公司 | Sperm quality multiparameter detection kit |
| CN116183470A (en) * | 2022-12-22 | 2023-05-30 | 中国林业科学研究院林业研究所 | Woody plant cell programmed death detection method based on flow sorting |
| CN116183470B (en) * | 2022-12-22 | 2024-04-05 | 中国林业科学研究院林业研究所 | Woody plant cell programmed death detection method based on flow sorting |
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