CN112304851A - Evaluation method of in vitro natural killer cell immunocompetence and application thereof - Google Patents
Evaluation method of in vitro natural killer cell immunocompetence and application thereof Download PDFInfo
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Abstract
The invention provides an evaluation method of in vitro natural killer cell immunocompetence and application thereof. The evaluation method comprises the following steps: mixing and co-culturing target cells carrying fluorescent markers A and natural killer cells, adding fluorescent markers B to mark the target cells and the natural killer cells, and adding fluorescent markers C to dye and mark dead cells generated after co-culturing; and then respectively using different fluorescence channels to carry out microscopic imaging on the cells, identifying and analyzing the cells in the same area in the microscopic images by combining an image synthesis analysis method, and evaluating the immunocompetence of the natural killer cells according to the analysis result. The evaluation method can directly obtain multiple data such as cell death rate, cell self-injury rate, cell specific killing rate and the like from the image, obtain the immunocompetence of the natural killer cell according to the data, and effectively eliminate the interference of impurities and cell fragments.
Description
Technical Field
The invention relates to the technical field of medical detection, and relates to an in vitro natural killer cell immunocompetence evaluation method and application thereof.
Background
Natural Killer (NK) cells are mainly present in peripheral blood, spleen and bone marrow, and are present in very small amounts in lymph nodes. It is different from T cell and B cell, and has no need of specific antibody to participate in killing target cell, and has no need of antigen to make pre-sensitization, and can quickly kill and dissolve several tumor cells or infected cells. Natural killer cells are closely related to the occurrence, development and treatment outcome of various diseases, so that the cellular immune function of the body can be understood to a greater extent through the evaluation of the immunological activity of natural killer cells.
The immunological activity of natural killer cells can be used as one of the indexes for judging the anti-tumor and anti-virus infection of organisms. In patients with hematological tumors, solid tumors, immunodeficiency, AIDS and certain viral infections, natural killer cell activity is reduced; in host versus graft responders, natural killer cell activity is elevated. In most patients with tumors, particularly in the mid-to-late stage and cancer with metastasis, the activity of natural killer cells in lymphocytes decreases and further decreases as the tumor progresses. After the operation of cancer patients, if the activity of natural killer cells is reduced continuously, the tumor is predicted to grow progressively or metastasize, and conversely, if the activity of natural killer cells is restored to normal, the treatment is predicted to be effective and the prognosis is good. In addition, the corresponding changes of the physiological functions of leukemia patients, organ transplantation patients and habitual abortion patients can be obviously reflected on the indexes of natural killer cell activity.
Therefore, the in vitro natural killer cell activity detection immunoassay has important guiding significance on the aspects of analysis, treatment monitoring, prognosis, outcome and the like of the etiology of tumors, blood diseases, infectious diseases, immune diseases, organ transplantation and the like.
The classical detection method of the killing effect of natural killer cells is a 51Cr release experiment, the method has good repeatability, but because the method uses isotopes to label target cells, multiple limiting factors such as short half-life period, high isotope waste treatment and experiment protection requirements and the like exist, and especially, the use of radioactive isotopes has great threat to health and environment, so that the application of the method is limited. In addition, the commonly used detection methods include Lactate Dehydrogenase (LDH) release method, Calcein release assay, and the like.
However, these detection methods are indirect methods, i.e., a certain reagent is used to label target cells, and then the target cells are incubated with natural killer cells of different concentrations, when the target cells are attacked and damaged by the natural killer cells, the permeability of cell membranes is changed, these labeled substances are released into the supernatant, and the activity of the natural killer cells is measured by measuring the released amount of the labeled substances; through indirect detection of a certain intermediate substance, the result is necessarily influenced by the intermediate substance, so that the unreliability of the detection result is increased; and the detection result is not visual, the accuracy of the detection result cannot be verified directly and repeatedly, and if the accuracy of the result needs to be verified, the evidence can be verified by observing with a microscope or other instruments.
Therefore, the method for evaluating the killing activity of the cells based on microscopic image recognition, intuition, visualization and accuracy is provided, and the method has important significance for evaluating the killing activity of the immune cells and developing the technology related to immunotherapy.
Disclosure of Invention
In view of the problems in the prior art, the invention provides an in vitro natural killer cell immunological activity evaluation method and application thereof. The method combines a fluorescence microscopic imaging method and an image synthesis analysis method, rapidly, intuitively and accurately counts the number of target cells and natural killer cells, and provides an evaluation result of the immunocompetence of the natural killer cells.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a method for evaluating natural killer cell immune activity in vitro, comprising the steps of:
(1) mixing and co-culturing target cells carrying fluorescent markers A and natural killer cells, adding fluorescent markers B to mark the target cells and the natural killer cells, and adding fluorescent markers C to dye and mark dead cells generated after co-culturing;
wherein the excitation light wavelengths corresponding to the fluorescent marker A, the fluorescent marker B and the fluorescent marker C are different;
(2) respectively carrying out microscopic imaging on the cells after the staining labeling by using a bright field, a fluorescence channel matched with the fluorescence label A, a fluorescence channel matched with the fluorescence label B and a fluorescence channel matched with the fluorescence label C to obtain a microscopic image;
(3) identifying cells in the obtained microscopic image through image identification, performing superposition synthesis analysis on the identification results of the same area, and evaluating the immunocompetence of the natural killer cells according to the analysis results;
in the process of the superposition synthesis analysis, cells which simultaneously display the fluorescent marker A and the fluorescent marker B are live target cells, cells which simultaneously display the fluorescent marker A, the fluorescent marker B and the fluorescent marker C are dead target cells, cells which only display the fluorescent marker B are live natural killer cells, and cells which simultaneously display the fluorescent marker B and the fluorescent marker C are dead natural killer cells.
In the invention, the tumor cells are marked by the fluorescent marker A to be used as target cells, the target cells and the natural killer cells are simultaneously marked by the fluorescent marker B, a proper effective target ratio is set, and the target cells and the natural killer cells are incubated together for a certain time; and then, labeling dead cells by using a fluorescent label C, putting the mixed sample into a counting plate, placing the counting plate under a microscopic fluorescence imaging system for shooting, and performing superposition synthesis analysis counting on microscopic images in the same view field, so that multiple data such as the killing rate of natural killer cells can be calculated, and the immunological activity of the natural killer cells can be further obtained.
In the invention, a fluorescent reagent A, a fluorescent marker B and a fluorescent marker C with three different excitation light wave bands are used for respectively dyeing and marking target cells, all cells and dead cells, then fluorescence microscopic imaging and image synthesis analysis are carried out, microscopic imaging is respectively carried out by using a bright field and a microscopic fluorescent channel suitable for 3 fluorescent markers at the same position or the same visual field, then microscopic images in the same visual field are superposed and synthesized and analyzed to obtain a detection result, namely, a direct reading method for directly obtaining the detection result from the images is adopted in the invention, and cadmium is compared51Release test andthe indirect method adopted by the flow cytometry is more accurate and visual in detection result.
Preferably, the fluorescent label A in step (1) comprises a fluorescent protein or a cellular dye.
The fluorescent protein includes any one of Green Fluorescent Protein (GFP), Red Fluorescent Protein (RFP) or luciferase (Luc).
In the present invention, the target cell may be various tumor cells corresponding to immune cells or virus-infected target cells. Target cells may carry a label such as GFP, RFP or Luc for identification, or may be labeled with a reactive dye such as CFSE (carboxyfluorescein diacetate succinimidyl ester) or Calcein AM (Calcein-AM).
Preferably, the step (1) further comprises washing after the target cells are stained and labeled with the fluorescent marker A.
Preferably, the target cells in step (1) include various tumor cells corresponding to immune cells and/or virus-infected cells.
For example, the target cell may BE K562 cell, Jurkat cell, neuroblastoma cell (e.g., SK-N-BE (2), CHLA155, or CHP 134) labeled with a fluorescent protein (e.g., GFP, RFP, or Luc), or other cells having the same mechanism of action as the aforementioned cells.
Preferably, the natural killer cells in step (1) comprise any one or a combination of at least two of human peripheral blood PBMC cells, NK cells obtained by separating and purifying from tissues or NK cells cultured by in vitro amplification; other cells having the same mechanism of action as the aforementioned cells may be used.
Preferably, the fluorescent label B in step (1) is a live cell dye.
In the present invention, when the target cell and the natural killer cell are stained together, the dye or the marker is preferably a nuclear dye, and may be a reagent that is different from the target cell dye and the dead cell dye. Preferably, the fluorescent label C in step (1) is a dead cell dye. The dead cell staining marker may be any dead cell staining marker dye, and may be, for example, any one of Annexin-V (Annexin-V), SYTOX Green (Cyanine SYTOX), PI (propylidine bromide), 7-AAD (7-amino actinomycin D), or a combination of at least two thereof. Alternatively, the fluorescent label C may be an agent that is distinguishable from the target cell dye.
In the present invention, the "live cell dye" and the "dead cell dye" refer to dyes capable of staining live cells and dead cells, respectively.
As a preferable embodiment of the present invention, when the target cells and the natural killer cells are co-cultured in step (1), the target cells and the natural killer cells are separately cultured as control groups.
Preferably, the evaluation method further comprises the steps of:
carrying out microscopic imaging on the contrast group by respectively using a bright field, a fluorescence channel matched with the fluorescence label A, a fluorescence channel matched with the fluorescence label B and a fluorescence channel matched with the fluorescence label C to obtain a microscopic image;
and carrying out image recognition on the microscopic image of the control group, and carrying out superposition synthesis analysis on the recognition result to obtain the target cell death rate of the control group and/or the natural killer cell death rate of the control group.
In a preferred embodiment of the present invention, the identification result in step (3) includes any one or a combination of at least two of position information, size information, and fluorescence intensity of the cell.
Preferably, the analysis result includes: the total number of target cells and natural killer cells, the number of live target cells, the number of dead target cells, the number of live natural killer cells, the number of dead natural killer cells, the death rate of target cells or the death rate of natural killer cells, or a combination of at least two of them.
Preferably, the method for evaluating in step (3) is:
and comparing the death rate of the target cells in the analysis result with a death rate threshold value, wherein the death rate threshold value comprises an upper limit and a lower limit, and obtaining the immunological activity of the natural killer cells according to the comparison result.
In the invention, if the death rate of the obtained target cells is more than or equal to the upper limit of the death rate threshold value, the evaluation of the immunocompetence of the natural killer cells is better; if the death rate of the target cell is less than the upper limit of the death rate threshold value but is more than or equal to the lower limit of the death rate threshold value, evaluating that the immunological activity of the natural killer cell is normal; if the resulting mortality of the target cells is less than the lower limit of the mortality threshold, the natural killer cells are assessed to be less immunocompetent.
In the invention, the fluorescent targets in the images of the fluorescent marker A, the fluorescent marker B and the fluorescent marker C are identified by a fluorescent image identification method to obtain the position information, the size information and the like of the detection target; and then, carrying out superposition synthesis analysis on the obtained microscopic image recognition results of the fluorescent marker A, the fluorescent marker B and the fluorescent marker C.
The position information and the size information of all cells can be acquired in the microscopic image of the fluorescent marker B; the position information and the size information of the living target cells can be simultaneously obtained in the microscopic image of the fluorescent marker A; the position information and the size information of dead target cells can be simultaneously obtained from microscopic images of the fluorescent marker A, the fluorescent marker B and the fluorescent marker C;
the position information and the size information of the living natural killer cells can be obtained only in the microscopic image of the fluorescent marker B; the positional information and the size information of dead natural killer cells can be simultaneously obtained in the microscopic images of the fluorescent marker B and the fluorescent marker C.
Specific information is shown in table 1:
TABLE 1
Wherein the target cell death rate is calculated using the following formula:
the target cell death rate is the number of dead target cells/(number of live target cells + number of dead target cells) × 100%;
the natural killer cell death rate is calculated by the following formula:
the natural killer cell death rate is equal to the number of dead natural killer cells/(number of live natural killer cells + number of dead natural killer cells) × 100%.
Similarly, if the target cell viability rate and the natural killer cell viability rate are calculated, the following formula is used:
the target cell survival rate is the number of live target cells/(number of live target cells + number of dead target cells) × 100%;
the natural killer cell survival rate is the number of living natural killer cells/(number of living natural killer cells + number of dead natural killer cells) × 100%.
Therefore, the sum of the death rate and the survival rate of the target cells is 100 percent, and the sum of the death rate and the survival rate of the natural killer cells is also 100 percent, which shows that various cell types are more accurate in the statistical process in the invention.
Preferably, the analysis result of step (3) further comprises: cell specific killing rate and/or natural killer cell self-injury rate.
Wherein, the specific killing rate of the cells is calculated by adopting the following formula:
cell-specific killing-target cell death-control target cell death;
the natural killer cell self-injury rate is calculated by adopting the following formula:
natural killer cell self-injury rate-effector cell death rate-control group natural killer cell death rate.
As a preferred technical solution of the present invention, the detection method comprises the steps of:
(1) mixing and co-culturing target cells carrying fluorescent markers A and natural killer cells, and simultaneously, independently culturing the target cells and the natural killer cells to be used as control cells;
the fluorescent marker A can be a fluorescent protein or a cell dye; the fluorescent protein label is transferred into a target cell through gene editing, so that the fluorescent protein label simultaneously expresses fluorescent protein in the growth process, and the commonly used fluorescent protein comprises any one of green fluorescent protein, red fluorescent protein or luciferase;
the fluorescent label A can also be a cell dye, if the selected cell dye is easy to generate background fluorescence, washing is carried out, and if the selected reagent has no background fluorescence or the background fluorescence does not influence the analysis, the step can be omitted;
(2) after the co-culture is finished, adding a fluorescent marker B to mark the target cells and the natural killer cells; the fluorescent marker B is a live cell dye and can stain all cells. Wherein the wavelength of the excitation light of the fluorescent marker B is different from that of the fluorescent marker A;
(3) then, using a fluorescent marker C to dye and mark dead cells generated after the co-culture;
the fluorescent marker C is a dead cell dye, wherein the wavelengths of exciting light corresponding to the fluorescent marker A, the fluorescent marker B and the fluorescent marker C are different; the fluorescent markers of three different exciting lights are selected to distinguish different cells, so that the subsequent statistical analysis is facilitated;
(4) transferring the stained cells to a cell counting plate or a cell counting plate with a structure similar to a micro-slit with a fixed distance with the cell counting plate;
(5) placing the counting plate which is prepared in the step (4) and is loaded with the sample under a micro-fluorescence imaging system for shooting, and respectively shooting microscopic images under a bright field channel, a fluorescence channel matched with a fluorescence label A, a fluorescence channel matched with a fluorescence label B and a fluorescence channel matched with a fluorescence label C at the same position or in the same field of view to obtain 4 micro-imaging pictures;
(6) image synthesis analysis counting: counting cells by using images obtained under a bright field channel and/or a fluorescence labeling B channel to obtain the total cell number; and then in 4 microscopic imaging pictures obtained by the superposition analysis, the cells simultaneously displaying the fluorescent marker A and the fluorescent marker B are live target cells, the cells simultaneously displaying the fluorescent marker A, the fluorescent marker B and the fluorescent marker C are dead target cells, the cells only displaying the fluorescent marker B are live natural killer cells, and the cells simultaneously displaying the fluorescent marker B and the fluorescent marker C are dead natural killer cells.
And finally, calculating the death rate of the target cells, the death rate of the natural killer cells, the death rates of the target cells and the natural killer cells of the control group, the specific cell killing rate and the self-injury rate of the natural killer cells, comparing the analysis result with a death rate threshold value, wherein the death rate threshold value comprises an upper limit and a lower limit, and obtaining the immunological activity of the natural killer cells according to the comparison result.
The calculation formula used is as follows:
the target cell death rate is the number of dead target cells/(number of live target cells + number of dead target cells) × 100%;
the natural killer cell death rate is equal to the number of dead natural killer cells/(the number of live natural killer cells + the number of dead natural killer cells) × 100%;
cell-specific killing (%) -target cell death-control target cell death;
natural killer cell self-injury (%) -effector cell death rate-control natural killer cell death rate.
In a second aspect, the present invention also provides a use of the detection method according to the first aspect for detecting natural killer cell immune activity, immune product quality control or individual immune function evaluation.
Compared with the prior art, the invention has at least the following beneficial effects:
(1) the invention provides a method for analyzing the immunocompetence of natural killer cells in vitro, which comprises the steps of respectively dyeing target cells, live cells and dead cells through three fluorescent labels with different wavelengths, obtaining different microscopic images under different wavelengths, distinguishing the live target cells, the dead target cells, the live natural killer cells and the dead natural killer cells through superposition synthesis analysis of the microscopic images, further obtaining the total number of the target cells and the natural killer cells, the number of the live target cells, the number of the dead target cells, the number of the live natural killer cells and the number of the dead natural killer cells, and calculating the results of cell killing rate and the like according to the corresponding cell numbers;
(2) the method provided by the invention combines fluorescence microscopic imaging and image synthesis analysis technology, can simultaneously obtain the image information and data processing result of the cell to be detected, and can count the number of various cells according to the fluorescence label compared with the detection result provided by a flow cytometer, so that the obtained result is more visual, and multiple data such as cell death rate, cell self-damage rate, cell specific killing rate and the like can be obtained on one instrument, thereby reducing the detection steps and improving the detection efficiency;
(3) the three-staining method provided by the invention judges all target cells and effector cells through fluorescent staining, and compared with a double-staining method for identifying in a bright field, impurities in the bright field can be removed more easily, and the result is more accurate; the double-staining method adopts the identification under the bright field as the total cell number, the impurities under the bright field are easy to be identified by mistake, and the three-staining method of the fluorescence method can eliminate the impurities under the bright field, effectively eliminate the interference of the impurities and cell fragments and enable the result to be more accurate.
Drawings
FIG. 1 is a fluorescence micrograph taken under the FL1 fluorescence channel of example 1.
FIG. 2 is a fluorescence micrograph taken under the FL2 fluorescence channel of example 1.
FIG. 3 is a fluorescence micrograph taken under the FL3 fluorescence channel of example 1.
FIG. 4 is a fluorescence microscopic image obtained by superimposing different fluorescence channels in example 1.
Detailed Description
The technical solutions of the present invention are further described in the following embodiments with reference to the drawings, but the following examples are only simple examples of the present invention and do not represent or limit the scope of the present invention, which is defined by the claims.
In the following examples, the reagents used are all available from conventional manufacturers; meanwhile, unless otherwise specified, the experimental methods used are conventional methods known to those skilled in the art.
Example 1
The embodiment provides an evaluation method of in vitro natural killer cell immunocompetence, which comprises the following specific steps:
(1) labeling of target cells:
culturing and collecting target cells, adding a fluorescent dye CFSE (purchased from Biolegend, USA) into the target cells for incubation, labeling, and preparing labeled target cells into a cell solution;
(2) preparing natural killer cells:
collecting early morning fasting venous blood, placing in an ethylenediamine tetraacetic acid (EDTA) anticoagulation sterile test tube, taking 3mL blood sample, mixing with 3mL normal saline, diluting, taking 4.5mL layering liquid
1077 to 15mL centrifuge tube, add 6mL diluted anticoagulated blood carefully along the tube wall onto the stratified fluid, keep the interface between the two clearly visible;
centrifuging at room temperature of 400g for 30min, separating into four layers in a centrifuge tube, wherein the top layer is plasma, the second layer is required PBMC cells, the third layer is separation liquid, the fourth layer is red blood cells, carefully sucking the PBMC cells of the second layer by using a 200-microliter gun head, and transferring the PBMC cells into a 1.5mL EP tube for later use;
(3) performing efficient target cell co-culture:
simultaneously adding target cells and natural killer cells into a culture dish, setting the effective-target ratio to be 10:1, sampling after incubation and co-culture are finished, adding Hoechst33342 (purchased from Thermofisiher, USA) and PI dye (purchased from Sigma, USA), and dyeing;
(4) adding the cells obtained in the step (3) into a blood counting chamber;
(5) placing the loaded blood count plate on a sample table of a detection instrument, and respectively taking a bright field channel, a FL1 channel (matched with a fluorescent dye Hoechst33342), a FL2 channel (matched with a fluorescent dye CFSE) and a FL3 channel (matched with a fluorescent dye PI);
wherein, the information and the sequence of the three fluorescence channels are respectively as follows:
FL1：Ex 375nm，Em 460nm；FL2：Ex 480nm，Em 535nm；FL3：Ex 525nm，Em 600LP；
the FL1 channel excites and collects Hoechst33342 fluorescence, the FL2 channel excites and collects CFSE fluorescence, and the FL3 channel excites and collects PI fluorescence.
(6) Then, microscopic imaging is carried out at the same position by using a bright field and a microscopic fluorescence channel suitable for 3 fluorescent test marks, and 4 microscopic imaging pictures are obtained;
(7) image synthesis analysis: the image recognition software will recognize the fluorescent pictures under the bright field, FL1 channel, FL2 channel, and FL3 channel:
wherein, the cells which simultaneously display Hoechst33342 fluorescence and CFSE fluorescence are live target cells, the cells which simultaneously display Hoechst33342 fluorescence, CFSE fluorescence and PI fluorescence are dead target cells, the cells which only display Hoechst33342 fluorescence are live natural killer cells, and the cells which simultaneously display Hoechst33342 fluorescence and PI fluorescence are dead natural killer cells;
the target cell mortality is calculated using the following formula:
the target cell death rate is the number of dead target cells/(number of live target cells + number of dead target cells) × 100%;
the natural killer cell death rate is calculated by the following formula:
the natural killer cell death rate is equal to the number of dead natural killer cells/(number of live natural killer cells + number of dead natural killer cells) × 100%.
The concrete correspondence is as follows:
the target of picture recognition under the FL1 channel is total cells (including target cells and natural killer cells);
the target of picture recognition under the FL2 channel is target cells (including live target cells and dead target cells);
pictures taken under the FL3 channel were identified as total dead cells (including dead target cells and dead natural killer cells);
after the images are superimposed, the results are shown in fig. 1 to 4, and the same positions where the cells are located are indicated. Fig. 1 is a microscopic image under the FL1 channel, fig. 2 is a microscopic image under the FL2 channel, fig. 3 is a microscopic image under the FL3 channel, and fig. 4 is a microscopic image obtained by superimposing the three channels.
If there is only FL1 signal, mark and count the number as a; here there are both FL1 and FL2 signals, denoted and counted as b; here there are both FL1 and FL3 signals, denoted and counted as c; here there are FL1, FL2 and FL3 signals at the same time, denoted and counted as d.
And self-defining an editing formula as follows:
b is the number of the living target cells; d represents the number of dead target cells;
target cell death (%) ═ d/(b + d) × 100;
the number of live natural killer cells is a; the number of dead natural killer cells is c;
the natural killer cell death rate (%) ═ c/(a + c) × 100.
(8) Comparing the mortality of the target cells with a mortality threshold value, wherein the mortality threshold value is set according to different situations, and the upper limit and the lower limit of the mortality threshold value are respectively set as 40% and 20%; it should be noted that the upper and lower mortality thresholds herein need to be evaluated in combination as a practical matter, and the numerical values herein are merely exemplary or referenced.
Among the obtained detection results:
in the experimental group with the death rate of the target cells being more than or equal to 40 percent, the natural killer cells have better immunocompetence;
in the experimental group with the death rate of the target cells being less than or equal to 20 percent, the natural killer cells have poor immune activity;
in the experimental group with the death rate of the target cells being more than 20% and less than 40%, the natural killer cells have normal immunological activity.
Example 2
This example provides a method for evaluating the immunological activity of natural killer cells in vitro, which is different from example 1 in that target cells and natural killer cells are cultured alone as a control group.
The method comprises the following specific steps:
(1) labeling of target cells:
culturing and collecting target cells, adding a fluorescent dye CFSE (purchased from Biolegend, USA) into the target cells for incubation, labeling, and preparing labeled target cells into a cell solution;
(2) preparing natural killer cells:
preparing natural killer cells into a cell solution;
(3) performing cell co-culture:
adding target cells and natural killer cells (as an experimental group) into a culture dish at the same time, setting the effective-to-target ratio to be 10:1, sampling after incubation and co-culture are finished, adding Hoechst33342 (purchased from Thermofisher, USA) and PI dye (purchased from Sigma, USA), and dyeing;
control cells were also prepared: only adding target cells and culture medium into a culture dish to serve as a target cell control group, and only adding natural killer cells and culture medium into another culture dish to serve as a natural killer cell control group;
culturing the control group and the experimental group in a culture environment;
(4) adding the cells obtained in the step (3) into a blood counting chamber;
(5) placing the loaded blood count plate on a sample table of a detection instrument, and respectively taking a bright field channel, a FL1 channel (matched with a fluorescent dye Hoechst33342), a FL2 channel (matched with a fluorescent dye CFSE) and a FL3 channel (matched with a fluorescent dye PI);
wherein, the information and the sequence of the three fluorescence channels are respectively as follows:
FL1：Ex 375nm，Em 460nm；FL2：Ex 480nm，Em 535nm；FL3：Ex 525nm，Em 600LP；
the FL1 channel excites and collects Hoechst33342 fluorescence, the FL2 channel excites and collects CFSE fluorescence, and the FL3 channel excites and collects PI fluorescence.
(6) Then, microscopic imaging is carried out at the same position by using a bright field and a microscopic fluorescence channel suitable for 3 fluorescent test marks, and 4 microscopic imaging pictures are obtained;
(7) image synthesis analysis: the image recognition software will recognize the fluorescent pictures under the bright field, FL1 channel, FL2 channel, and FL3 channel:
after the images are superimposed, the software marks the same position where the cell is located:
if there is only FL1 signal, mark and count the number as a; here there are both FL1 and FL2 signals, denoted and counted as b; here there are both FL1 and FL3 signals, denoted and counted as c; here there are FL1, FL2 and FL3 signals at the same time, denoted and counted as d.
And self-defining an editing formula as follows:
b is the number of the living target cells; d represents the number of dead target cells;
target cell death (%) ═ d/(b + d) × 100;
the number of live natural killer cells is a; the number of dead natural killer cells is c;
the natural killer cell death rate (%) ═ c/(a + c) × 100.
In addition to obtaining the target cell death rate and the natural killer cell death rate of the experimental group, the target cell death rate and the natural killer cell death rate of the control group can be obtained in the embodiment, so that the cell specific killing rate and the natural killer cell self-injury rate can be calculated;
wherein, the specific killing rate (%) of the cells is target cell death rate-target cell death rate of the control group; natural killer cell self-injury (%) -effector cell death rate-control natural killer cell death rate.
Under the condition of setting a contrast, the influence of the conditions of cell natural death and the like on the result of the evaluation of the immune activity of the natural killer cells is eliminated, and the detection accuracy is improved.
In conclusion, the method provided by the invention is visual, and can directly obtain a visual cell fluorescence image, and different states of cells are obtained based on a microscopic image so as to analyze the killing effect of immune cells. The method has simple steps and improves the detection efficiency and accuracy.
The applicant declares that the above description is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be understood by those skilled in the art that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are within the scope and disclosure of the present invention.
Claims (10)
1. An in vitro natural killer cell immune activity evaluation method, which is characterized by comprising the following steps:
(1) mixing and co-culturing target cells carrying fluorescent markers A and natural killer cells, adding fluorescent markers B to mark the target cells and the natural killer cells, and adding fluorescent markers C to dye and mark dead cells generated after co-culturing;
wherein the excitation light wavelengths corresponding to the fluorescent marker A, the fluorescent marker B and the fluorescent marker C are different;
(2) respectively carrying out microscopic imaging on the cells after the staining labeling by using a bright field, a fluorescence channel matched with the fluorescence label A, a fluorescence channel matched with the fluorescence label B and a fluorescence channel matched with the fluorescence label C to obtain a microscopic image;
(3) identifying cells in the obtained microscopic image through image identification, performing superposition synthesis analysis on the identification results of the same area, and evaluating the immunocompetence of the natural killer cells according to the analysis results;
in the process of the superposition synthesis analysis, cells which simultaneously display the fluorescent marker A and the fluorescent marker B are live target cells, cells which simultaneously display the fluorescent marker A, the fluorescent marker B and the fluorescent marker C are dead target cells, cells which only display the fluorescent marker B are live natural killer cells, and cells which simultaneously display the fluorescent marker B and the fluorescent marker C are dead natural killer cells.
2. The method of claim 1, wherein the fluorescent marker A in step (1) comprises a fluorescent protein or a cellular dye;
preferably, the fluorescent protein comprises any one of green fluorescent protein, red fluorescent protein or luciferase;
preferably, after the target cells are stained and labeled by using the fluorescent marker A in the step (1), the washing operation is also included;
preferably, the fluorescent marker B in step (1) is a live cell dye;
preferably, the fluorescent label C in step (1) is a dead cell dye.
3. The method of claim 1 or 2, wherein the target cells in step (1) comprise virus-infected cells and/or immune cell-targeted tumor cells;
preferably, the target cells in step (1) comprise tumor cells targeted by immune cells and/or virus-infected cells;
preferably, the target cells in step (1) comprise any one or a combination of at least two of K562 cells, Jurkat cells or neuroblastoma cells without or with a fluorescent protein marker;
preferably, the natural killer cells in step (1) comprise any one of or a combination of at least two of human peripheral blood PBMC cells, NK cells obtained by isolation and purification from tissues, or NK cells cultured by in vitro amplification.
4. The method according to any one of claims 1 to 3, wherein the target cells and the natural killer cells are separately cultured as a control group when the target cells and the natural killer cells are co-cultured in step (1).
5. The evaluation method according to claim 4, characterized in that the evaluation method further comprises the steps of:
carrying out microscopic imaging on the contrast group by respectively using a bright field, a fluorescence channel matched with the fluorescence label A, a fluorescence channel matched with the fluorescence label B and a fluorescence channel matched with the fluorescence label C to obtain a microscopic image;
and carrying out image recognition on the microscopic image of the control group, and carrying out superposition synthesis analysis on the recognition result to obtain the target cell death rate of the control group and/or the natural killer cell death rate of the control group.
6. The method according to any one of claims 1 to 5, wherein the identification result in step (3) includes any one of or a combination of at least two of position information, size information, and fluorescence intensity of the cell.
7. The evaluation method according to any one of claims 1 to 6, wherein the analysis result of step (3) includes: any one or combination of at least two of total number of target cells and natural killer cells, number of living target cells, number of dead target cells, number of living natural killer cells, number of dead natural killer cells, target cell death rate or natural killer cell death rate;
preferably, the method for evaluating in step (3) is:
comparing the mortality rate of the target cells in the analysis result with a mortality threshold value, wherein the mortality threshold value comprises an upper limit and a lower limit, and obtaining the immunocompetence level of the natural killer cells according to the comparison result.
8. The evaluation method according to claim 7, wherein the target cell death rate is calculated using the following formula:
the target cell death rate is the number of dead target cells/(number of live target cells + number of dead target cells) × 100%;
preferably, the natural killer cell death rate is calculated using the following formula:
the natural killer cell death rate is equal to the number of dead natural killer cells/(number of live natural killer cells + number of dead natural killer cells) × 100%.
9. The evaluation method according to any one of claims 1 to 8, wherein the analysis result of step (3) further comprises: cell specific killing rate and/or natural killer cell self-injury rate;
preferably, the cell-specific killing rate is calculated using the following formula:
cell-specific killing-target cell death-control target cell death;
preferably, the natural killer cell self-injury rate is calculated by using the following formula:
natural killer cell self-injury rate-effector cell death rate-control group natural killer cell death rate.
10. Use of the method of any one of claims 1 to 9 for detecting natural killer cell immune activity, immune product quality control or individual immune function evaluation.
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| EP21885279.6A EP4212853A4 (en) | 2020-10-28 | 2021-10-28 | Method and system for detecting cell killing efficacy and/or immune activity, and application thereof |
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