Detection method and kit for killing efficiency of NK (Natural Killer) cells

Abstract

The invention discloses a detection method for killing efficiency of NK cells and a kit. The detection method comprises the following steps: S1, preparing a target cell suspension and an NK cell suspension; s2, setting experiment groups including a standard curve group, an experiment group and a blank group; the cells are inoculated to two 96-well plates respectively, each 96-well plate is provided with three multiple holes, and inoculation is carried out according to experimental groups; s3, respectively adding a CCK-8 reagent into the experiment holes of a 96-well plate, and measuring the absorbance of each hole at the position of 450 nm through a microplate reader; s4, placing another 96-well plate in the incubator for continuous co-culture for a certain period of time, respectively adding a CCK-8 reagent into each experiment hole, and measuring the absorbance of each hole at the position of 450 nm through the microplate reader; and S5, carrying out regression analysis on the OD value and the cell number of the standard curve group, and if R2 is greater than or equal to 0.9, calculating the killing rate according to a formula. According to the detection method and the kit for the killing efficiency of the NK cells, the killing activity of the NK cells on K562 and A549 is detected by applying CCK-8, the detection method for detecting the killing tumor cells of the NK cells through a CCK-8 method is established, operation is simpler and more convenient, consumed time is short, repeatability is good, damage to the cells is small, and the result is accurate.

Description

Technical Field

[0001] This invention relates to the technical field of cell killing efficiency detection, specifically a method and kit for detecting NK cell killing efficiency. Background Technology

[0002] Natural killer (NK) cells originate from lymphoprogenitor cells in the bone marrow and mature in the bone marrow or thymus. They are involved not only in anti-tumor, antiviral, and immune regulation but also in the development of hypersensitivity reactions and autoimmune diseases, making them an important component of the innate immune system. NK cells are classified into CD56 and CD16 based on the difference in their expression. bright CD16 - and CD56 dim CD16 + Two main subgroups, CD56 bright CD16 - NK cells lack the expression of killer cell immunoglobulin-like receptors but can express a large number of cytokines, such as interferon-γ (IFN-γ), interleukin-10 (IL-10), and tumor necrosis factor-α (TNF-α). CD4+, on the other hand, lack the expression of these receptors. dim CD16 + NK cells contain high levels of perforin, granzymes, and cytolytic particles, express cytotoxic cell immunoglobulin-like receptors, and exhibit high cytotoxicity. Perforin, granzymes, and cytokines produced during NK cell activation can promote tumor cell apoptosis, thus exerting an anti-tumor effect. Based on their unique anti-tumor properties, various treatment strategies have been developed, including cytokine therapy, monoclonal antibodies, NK cell infusion, and CAR-NK, demonstrating broad prospects for clinical application.

[0003] Currently, traditional detection methods include flow cytometry, isotope release assays, and lactate dehydrogenase (LDH) release assays. Flow cytometry, as the mainstream method, is widely used but has significant drawbacks. This method requires fluorescent labeling of target cells, followed by a complex cell collection process after co-culturing. Especially when the target cells are adherent cells, digestion is necessary, and these repeated pipetting and centrifugation processes can easily cause cell damage, severely affecting the accuracy of experimental results.

[0004] Furthermore, while isotope release methods offer high detection sensitivity, they suffer from drawbacks such as radioactive hazards, high reagent costs, cumbersome operation, and the need for specialized protective equipment, severely limiting their application in routine laboratory environments. In addition, the lactate dehydrogenase (LDH) release method, based on the principle of LDH enzyme release following cell membrane damage, suffers from low sensitivity, susceptibility to interference from serum components in the culture medium, narrow linear detection range, and poor repeatability, making it difficult to meet the demands for precise detection. Therefore, this application provides a solution. Summary of the Invention

[0005] This application provides a method and kit for detecting NK cell killing efficiency. By applying CCK-8 to detect the killing activity of NK cells against K562 and A549, a CCK-8 method for detecting NK cell killing of tumor cells is established. This method is simpler to operate, less time-consuming, has good repeatability, causes less cell damage, and provides accurate results. The required reagents are also cheaper and safer.

[0006] This application provides a method for detecting NK cell killing efficiency, characterized by the following steps: S1, preparing target cell suspension and NK cell suspension, wherein the target cells are selected from K562 cells or A549 cells; S2, setting up experimental groups, including a standard curve group, an experimental group, and a blank group; seeding cells onto two 96-well plates, each 96-well plate having three replicates per group, and seeding according to the experimental groups, adding NK cell suspension and target cell suspension to the experimental group; adding the corresponding NK cell suspension or target cell suspension to the standard curve group; adding culture medium to the blank group; S3, adding CCK-8 reagent to each replicate well of a 96-well plate, incubating in an incubator for a certain time, and measuring the 450 nm of each replicate well using a microplate reader. The absorbance at 450 nm was measured to obtain the following data: OD1 of the standard curve group, OD1 of the experimental group, and OD1 of the blank group; S4. Another 96-well plate was placed in an incubator for further co-incubation. After the experimental endpoint, CCK-8 reagent was added to each replicate well of the 96-well plate, and the plate was incubated for a certain period of time. The absorbance at 450 nm of each replicate well was then measured using a microplate reader to obtain the following data: OD2 of the standard curve group, OD2 of the experimental group, and OD2 of the blank group; S5. The regression equation R for each standard curve group was calculated. 2 If R 2 If the value is ≥0.9, the kill rate is calculated according to the formula.

[0007] By adopting the above technical solution, this application presents a detection method based on the CCK-8 assay. Through optimization of parameters (such as the effector-to-target ratio, co-culture time, and standard curve), a simple, safe, and highly sensitive method for detecting NK cell killing efficiency is achieved. Currently, most methods for detecting NK cell killing efficiency are flow cytometry. However, this method requires tracer treatment of target cells, collection of co-cultured cells after co-culture, and digestion if the target cells are adherent. These processes, along with the flow cytometry pretreatment, require repeated pipetting and centrifugation, which can easily cause cell damage and lead to inaccurate results. Therefore, the CCK-8 assay is superior to flow cytometry for detecting NK cell killing activity.

[0008] In the assay of NK cell cytotoxic activity, different culture batches of NK cells and target cells, as well as cell states, can affect the cytotoxic efficiency. Therefore, we established a standard curve between cell quantity and OD value to assess its linear correlation, using this as a quality control point to demonstrate the reliability of the experiment. The experimental results demonstrate the application and reliability of the CCK-8 assay in detecting NK cell cytotoxic activity.

[0009] Preferably, the standard curve concentration of the target cells is 1.6 × 10⁻⁶. 5 cells / mL, 1.4 × 10 5 cells / mL, 1.2×10 5 cells / mL, 1.0×10 5 cells / mL, 0.8×10 5 cells / mL, 0.6×10 5 cells / mL, 0.4×10 5 cells / mL, 0.2×10 5 cells / mL; the standard curve cell concentration for NK cells was 30 × 10⁻⁶. 5 cells / mL, 25×10 5 cells / mL, 20×10 5 cells / mL, 15×10 5 cells / mL, 10×10 5 cells / mL, 5×10 5 cells / mL, 1.0×10 5 cells / mL, 0.2×10 5 cells / mL.

[0010] By employing the above technical solution, cell activity is quantified using a standard curve, improving accuracy and reducing background interference. Specifically, establishing a standard curve concentration for target cells ensures linearity across the entire range, improves the sensitivity for detecting low-activity cells, and expands the NK cell concentration range to accommodate NK cells in different amplification states, ensuring accurate detection even at high cell counts.

[0011] Preferably, the target cell concentration in the experimental group is 0.5 × 10⁻⁶. 5 cells / mL; the effector-to-target ratios of the experimental groups were 10:1, 50:1, and 100:1; the NK cell concentrations were 5 × 10⁻⁶ cells / mL. 5 cells / mL, 25×10 5 cells / mL, 50×10 5cells / mL. The experimental groups were set with effect-to-target ratios of 10:1, 50:1, and 100:1.

[0012] Preferably, in step S5, the formula for calculating the lethality is: lethality = [(experimental group OD1 - blank group OD1) - (experimental group OD2 - blank group OD2)] / (experimental group OD1 - blank group OD1) × 100%.

[0013] Preferably, the method further includes the resuscitation and culture of target cells; the resuscitation and culture of target cells includes the resuscitation and culture of K562 cells and the resuscitation and culture of A549 cells; the resuscitation and culture of K562 cells includes the following steps: S1, transfer the resuscitated and frozen K562 cells to complete culture medium, mix by pipetting, centrifuge and discard the supernatant, resuspend in complete culture medium, and count the cells using an AO / PI cell counter; S2, add complete culture medium to the cell culture flask, seed K562 cells into the culture flask, and place in an incubator for static culture. When the cell confluence is 80%-90%, pass the cells or freeze them.

[0014] Preferably, the resuscitation and culture of A549 cells includes the following steps: S1, resuscitate frozen A549 cells and transfer them to complete culture medium, mix by pipetting, centrifuge, discard the supernatant, resuspend in complete culture medium, and count cells using an AO / PI cell counter; S2, add complete culture medium to a cell culture flask, seed A549 cells into the flask, and place it in an incubator for static culture; S3, when the cell confluence is 80%-90%, passage or cryopreserve the cells, remove the culture flask, discard the supernatant, add cell digestive enzymes for digestion, observe the cells become rounded, add buffer to stop digestion, collect the cell suspension, centrifuge, discard the supernatant, resuspend in complete culture medium, and count cells using an AO / PI cell counter.

[0015] On the other hand, this application discloses a kit for detecting NK cell killing efficiency, including CCK-8 reagent, target cell culture reagent and FBS, wherein the target cell culture reagent is selected from RPMI 1640 medium and Ham's F-12K medium.

[0016] Preferably, it also includes a standard curve concentration gradient reference card for establishing a standard curve, the reference card being printed with a target cell concentration gradient of 0.2 × 10⁻⁶. 5 cells / mL up to 1.6 × 10⁻⁶ 5 cells / mL and NK cell concentration gradient 0.2×10 5 cells / mL up to 30 × 10 5 cells / mL.

[0017] By adopting the above technical solution, this application productizes the method, which facilitates standardized application, lowers the user's operating threshold, and enables rapid location of concentration gradients through visual guidance, thereby reducing calculation errors.

[0018] One or more technical solutions provided in this application have at least the following technical effects or advantages: 1. This application uses a detection method based on the CCK-8 assay. By optimizing parameters (such as the effector-to-target ratio, co-culture time, and standard curve), it achieves a simple, safe, and highly sensitive detection of NK cell killing efficiency. By establishing a standard curve between cell quantity and OD value, the linear correlation is evaluated, which serves as a quality control point to prove the reliability of the experiment.

[0019] 2. This application quantifies cell activity using a standard curve, improving accuracy and reducing background interference. Establishing a standard curve for target cell concentration ensures linearity across the entire range, improves sensitivity for low-activity detection, and expands the NK cell concentration range to accommodate NK cells in different amplification states, ensuring accurate detection even at high cell counts.

[0020] 3. This application productizes the method, which facilitates standardized application, lowers the user's operating threshold, and provides visual guidance to quickly locate the concentration gradient and reduce calculation errors. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a standard curve of K562 cells at 0h in Example 1 of this application; Figure 2 This is a standard curve of NK cells at 0h in Example 1 of this application; Figure 3 This is a standard curve of K562 cells at 4 hours in Example 1 of this application; Figure 4 This is a standard curve of NK cells at 4 hours in Example 1 of this application; Figure 5 This is a statistical chart showing the killing rate of NK cells against K562 cells in Example 1 of this application; Figure 6 This is a standard curve of A549 cells at 0h in Example 1 of this application; Figure 7 This is a standard curve of NK cells at 0h in Example 1 of this application; Figure 8 This is a standard curve of A549 cells at 4 hours in Example 1 of this application; Figure 9 This is a standard curve of NK cells at 4 hours in Example 1 of this application; Figure 10 This is a statistical chart showing the killing rate of NK cells against A549 cells in Example 1 of this application. Detailed Implementation

[0023] This application provides a method and kit for detecting NK cell killing efficiency. By applying CCK-8 to detect the killing activity of NK cells against K562 and A549, a CCK-8 method for detecting NK cell killing of tumor cells is established. This method is simpler to operate, less time-consuming, has good repeatability, causes less cell damage, and provides accurate results. The required reagents are also cheaper and safer.

[0024] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0025] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this application are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of this application described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion; for example, a process, method, system, product, or server that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or modules not explicitly listed or inherent to such processes, methods, products, or devices.

[0026] First, this application discloses a method for detecting NK cell killing efficiency, specifically including the resuscitation and culture of target cells and the NK cell killing efficiency test.

[0027] Furthermore, the resuscitation and culture of target cells includes the resuscitation and culture of K562 cells and A549 cells.

[0028] Specifically, the resuscitation and culture of K562 cells includes the following steps: S1. Resuscitate the frozen K562 cells and quickly transfer them to RPMI 1640 complete medium (containing 10% FBS). Mix well by pipetting, centrifuge and discard the supernatant. Resuspend the cells in RPMI 1640 complete medium and count the cells using an AO / PI cell counter.

[0029] Add 15 mL of RPMI 1640 complete culture medium to S2 and T75 cell culture flasks, and take 3.0 × 10⁻⁶ cells. 6 Seed K562 cells into T75 culture flasks and gently shake to disperse the cells evenly. Incubate statically at 37°C and 5% CO2. Passage or cryopreserve the cells when they reach 80%-90% confluence.

[0030] The resuscitation and culture of A549 cells includes the following steps: S1. Resuscitate the frozen A549 cells and quickly transfer them to Ham's F-12K complete medium (containing 10% FBS). Mix well by pipetting, centrifuge, discard the supernatant, and resuspend in Ham's F-12K complete medium. Count the cells using an AO / PI cell counter.

[0031] S2. Add 15 mL of Ham's F-12K complete medium to a T75 cell culture flask, and take 7.5 × 10⁻⁶ cells. 5 One A549 cell was seeded into a T75 culture flask and gently shaken to disperse the cells evenly. The flask was then placed in an incubator for static culture at 37°C and 5% CO2.

[0032] S3. When the cell confluence reaches 80%-90%, passage or cryopreserve the cells. For passage, remove the culture flask, discard the supernatant, add 3 mL of Tryple for digestion for 5 min, observe under a microscope that the cells have become rounded, add 10 mL of PBS to stop the digestion, gently pipette, collect the cell suspension, centrifuge, discard the supernatant, resuspend in Ham's F-12K complete medium, and count the cells using an AO / PI cell counter.

[0033] Furthermore, the killing efficiency of NK cells was tested, including the following steps: S1. Prepare target cell suspension and NK cell suspension, wherein the target cells are selected from K562 cells or A549 cells; S2. Set up experimental groups, including standard curve group, experimental group and blank group; seed cells into two 96-well plates, each with three replicates, and seed according to the experimental groups. Add NK cell and target cell suspensions to the experimental group; add the corresponding NK cell suspension or target cell suspension to the standard curve group; add complete culture medium to the blank group. S3. Add CCK-8 reagent to each replicate well of a 96-well plate and incubate in an incubator for a certain period of time. Measure the absorbance at 450 nm of each replicate well using a microplate reader to obtain the following data: OD1 of the standard curve group, OD1 of the experimental group, and OD1 of the blank group. S4. Place another 96-well plate in an incubator and continue co-culturing. After the experimental endpoint, add CCK-8 reagent to each replicate well of the 96-well plate and incubate in an incubator for a certain period of time. Then, measure the absorbance at 450 nm of each replicate well using an ELISA reader to obtain the following data: OD2 of the standard curve group, OD2 of the experimental group, and OD2 of the blank group. S5. Perform regression analysis on the standard curve group OD1, OD2 and cell count. If R 2 If the value is ≥0.9, the kill rate is calculated according to the formula.

[0034] Specifically, the embodiments of this application test the killing efficiency of NK cells based on different target cells. Example 1

[0035] Example 1 is a test of the cytotoxic activity of NK cells against K562 cells. The specific steps are as follows: S1. Prepare the K562 target cell suspension cultured above; then, centrifuge the cultured NK cells, discard the supernatant, and resuspend them in the corresponding RPMI 1640 complete medium.

[0036] S2. Set up experimental groups, including a standard curve group, an experimental group, and a blank group. Prepare K562 and NK cell suspensions at different concentrations according to the experimental groups and effector-target ratio.

[0037] The experiment was then divided into groups, including the K562 cell standard curve group, the NK cell standard curve group, the experimental group (effect-to-target ratio of 10:1, 50:1, and 100:1, respectively), and the blank group.

[0038] Specifically, cells were seeded onto two 96-well plates, with three replicates per well on each plate. Cells were seeded into each 96-well plate according to the experimental groups. 50 μL of NK cell suspension and 50 μL of K562 cell suspension were added to each experimental group; 100 μL of NK cell suspension was added to each NK cell standard curve group; 100 μL of K562 cell suspension was added to each K562 standard curve group; and 100 μL of complete culture medium was added to each blank group.

[0039] S3. Add 10 μL of CCK-8 reagent to each replicate well of a 96-well plate and incubate at 37°C with 5% CO2 for 1.5 h. Measure the absorbance at 450 nm in each replicate well using a microplate reader to obtain the following data: OD1 of NK cell standard curve group, OD1 of K562 cell standard curve group, OD1 of experimental group, and OD1 of blank group. S4. Place another 96-well plate in a 37℃, 5% CO2 incubator and co-culture for 4 hours. After the experimental endpoint, add 10 μL of CCK-8 reagent to each replicate well of the 96-well plate and incubate in a CO2 incubator for 1.5 hours. Then, measure the absorbance at 450 nm of each replicate well using an ELISA reader to obtain the following data: OD2 of NK cell standard curve group, OD2 of K562 cell standard curve group, OD2 of experimental group, and OD2 of blank group. S5. Regression analysis was performed on the OD1 and OD2 of NK cells in the standard curve group and the OD1 and OD2 of K562 cells. If R 2 If the result is ≥0.9, the kill rate is calculated using the formula: Kill rate = [(Experimental group OD1 - Blank group OD1) - (Experimental group OD2 - Blank group OD2)] / (Experimental group OD1 - Blank group OD1) × 100%.

[0040] Reference Figure 1 The figure shows the standard curve of K562 cells at 0h obtained in Example 1 of this application, illustrating the linear relationship between different numbers of K562 cells and OD values ​​at the experimental starting point. The curve slope is stable and the R² value is high, indicating uniform cell activity, small systematic error, and a linear correlation between cell quantity and OD value, providing a reliable benchmark for subsequent quantification. Figure 2 The standard curve of NK cells at 0h obtained in Example 1 of this application was used to establish the activity baseline of NK cells themselves, which was used to evaluate the correlation between NK cell quantity and OD value. Figure 3 This is the standard curve of K562 cells obtained in Example 1 of this application at 4 hours. Figure 4 This is a standard curve of NK cells at 4 hours obtained in Example 1 of this application. (And combined with...) Figure 5 analyze, Figure 5 The graph shows the cytotoxicity of NK cells against K562 cells, illustrating the cytotoxicity at different effector-to-target ratios. The data indicates that the cytotoxicity significantly increases as the effector-to-target ratio increases from 10:1 to 100:1, demonstrating a positive correlation between NK cell cytotoxic activity and the effector-to-target ratio. Example 2

[0041] The difference between Example 2 and Example 1 is that the target cells used in Example 2 are A549. Cells, the specific steps are as follows: S1. Prepare the suspension of the cultured A549 cells; then, centrifuge the cultured NK cells, discard the supernatant, and resuspend them in Ham's F-12K complete medium.

[0042] S2. Set up experimental groups, including a standard curve group, an experimental group, and a blank group. Prepare A549 and NK cell suspensions at different concentrations according to the experimental groups and effector-target ratio.

[0043] Then, the experiments were divided into groups, including the A549 cell standard curve group, the NK cell standard curve group, the experimental group (effect-to-target ratios of 10:1, 50:1, and 100:1, respectively), and the blank group.

[0044] Specifically, cells were seeded onto two 96-well plates, with three replicates per well on each plate. Cells were seeded into each 96-well plate according to the experimental groups. 50 μL of NK cell suspension and 50 μL of A549 cell suspension were added to each experimental group; 100 μL of NK cell suspension was added to each NK cell standard curve group; 100 μL of A549 cell suspension was added to each A549 cell standard curve group; and 100 μL of complete culture medium was added to each blank group.

[0045] S3. Add 10 μL of CCK-8 reagent to each replicate well of a 96-well plate and incubate at 37°C with 5% CO2 for 1.5 h. Measure the absorbance at 450 nm for each replicate well using a microplate reader to obtain the following data: OD1 of NK cell standard curve group, OD1 of A549 cell standard curve group, OD1 of experimental group, and OD1 of blank group. S4. Another 96-well plate was placed in a 37℃, 5% CO2 incubator and co-cultured for 4 hours. After the experimental endpoint, 10 μL of CCK-8 reagent was added to each replicate well of the 96-well plate and incubated in a CO2 incubator for 1.5 hours. The absorbance at 450 nm of each replicate well was then measured using an ELISA reader. The data obtained were: OD2 of NK cell standard curve group, OD2 of A549 cell standard curve group, OD2 of experimental group, and OD2 of blank group. S5. Regression analysis was performed on the OD1 and OD2 of NK cells in the standard curve group and the OD1 and OD2 of A549 cells. If R 2 If the result is ≥0.9, the kill rate is calculated using the formula: Kill rate = [(Experimental group OD1 - Blank group OD1) - (Experimental group OD2 - Blank group OD2)] / (Experimental group OD1 - Blank group OD1) × 100%.

[0046] Reference Figure 6 The graph shown is a standard curve of A549 cells at 0h obtained in Example 2 of this application, illustrating the linear relationship between the number of A549 cells and OD at 0h. Figure 7 This is the standard curve of NK cells at 0h obtained in Example 2 of this application, compared with... Figure 2 Similarly, it provides baseline correction for NK cells.

[0047] in addition, Figure 8 This is the standard curve of A549 cells obtained in Example 2 of this application at 4 hours. Figure 9This is the standard curve of NK cells at 4 hours obtained in Example 2 of this application. 2 All values ​​were greater than 0.9, and the linear relationship between cell number and OD value was good, indicating that the cells were in good growth condition during the experimental period and the systematic error was small, suggesting that the method is also stable for adherent cells. and combined Figure 10 analyze, Figure 10 The graph shows the cytotoxicity of NK cells against A549 cells. It indicates that the cytotoxicity significantly increases with the increase of the effector-to-target ratio. The cytotoxicity is almost the same when the effector-to-target ratio is 50:1 and 100:1, suggesting that the cytotoxicity of this batch of NK cells against A549 cells may reach its maximum at a ratio of 50:1.

[0048] In summary, all standard curves ( Figures 1-4 , Figures 6-9 All results showed good linearity (R² > 0.9), ensuring that the OD values ​​in each experiment reliably reflected the cell number, thus improving the reliability and accuracy of the data. Furthermore, Figure 5 and Figure 10 The results showed that the killing rate increased with the increase of the effector-to-target ratio, and the results were statistically significant (p < 0.05 or p < 0.01), demonstrating that the method can sensitively reflect the dose-response relationship of NK cell killing activity and is superior to the traditional method.

[0049] Furthermore, this application uses CCK-8 reagent for one-step addition, eliminating the need for complex processing (such as labeling and centrifugation in flow cytometry). Moreover, this application causes minimal cell damage, directly detecting metabolic activity and avoiding physical damage (especially for adherent cells).

[0050] In addition, this application provides a kit for detecting NK cell killing efficiency, including CCK-8 reagent and target cell culture reagent, and also includes a standard curve concentration gradient reference card for establishing a standard curve. The reference card contains a target cell concentration gradient of 0.2 × 10⁻⁶. 5 cells / mL up to 1.6 × 10⁻⁶ 5 cells / mL and NK cell concentration gradient 0.2×10 5 cells / mL up to 30 × 10 5 cells / mL. Additionally, the target cell culture reagents were selected from RPMI 1640 medium and Ham's F-12K medium, and the serum required for cell culture was premium fetal bovine serum.

[0051] This application commercializes the method, facilitating standardized application, lowering the operational threshold for users, and providing visual guidance for rapid location of concentration gradients, reducing calculation errors. Furthermore, this application's solution is low-cost, uses inexpensive reagents, and requires no radioactive or specialized equipment.

[0052] It should be noted that the order of the embodiments described above is merely for descriptive purposes and does not represent the superiority or inferiority of the embodiments. Furthermore, specific embodiments have been described above. Other embodiments are within the scope of the appended claims. In some cases, the actions or steps described in the claims can be performed in a different order than that shown in the embodiments and still achieve the desired result. Additionally, the processes depicted in the drawings do not necessarily require a specific or sequential order to achieve the desired result. In some embodiments, multitasking and parallel processing are also possible or may be advantageous.

[0053] The above description is only a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

[0054] This specification and accompanying drawings are merely illustrative examples of this application and are intended to cover any and all modifications, variations, combinations, or equivalents within the scope of this application. Clearly, those skilled in the art can make various alterations and modifications to this application without departing from its scope. Therefore, if such modifications and modifications fall within the scope of this application and its equivalents, this application intends to include such modifications and modifications.

Claims

1. A method for detecting NK cell killing efficiency, characterized in that, Includes the following steps: S1. Prepare target cell suspension and NK cell suspension, wherein the target cells are selected from K562 cells or A549 cells; S2. Set up experimental groups, including standard curve group, experimental group and blank group; seed cells into two 96-well plates, with three replicates for each group, and seed according to the experimental groups. Add NK cell and target cell suspensions to the experimental group; add the corresponding NK cell suspension or target cell suspension to the standard curve group; add complete culture medium to the blank group. S3. Add CCK-8 reagent to each well of a 96-well plate and incubate in an incubator for a certain period of time. Then, measure the absorbance at 450 nm of each well using a microplate reader to obtain the following data: OD1 of the standard curve group, OD1 of the experimental group, and OD1 of the blank group. S4. Place another 96-well plate in the incubator and continue co-culturing. After the experimental endpoint, add CCK-8 reagent to each well of the 96-well plate and incubate in the incubator for a certain time. Measure the absorbance at 450 nm of each well using an ELISA reader to obtain the following data: OD2 of the standard curve group, OD2 of the experimental group, and OD2 of the blank group. S5. Perform regression analysis on the standard curve sets OD1 and OD2. If R 2 If the value is ≥0.9, the kill rate is calculated according to the formula.

2. The method for detecting NK cell killing efficiency as described in claim 1, characterized in that, The standard curve concentrations of the target cells were 1.6 × 10⁻⁶. 5 cells / mL, 1.4 × 10 5 cells / mL, 1.2×10 5 cells / mL, 1.0×10 5 cells / mL, 0.8×10 5 cells / mL, 0.6×10 5 cells / mL, 0.4×10 5 cells / mL, 0.2×10 5 cells / mL; The standard curve for NK cells showed cell concentrations of 30 × 10⁻⁶. 5 cells / mL, 25×10 5 cells / mL, 20×10 5 cells / mL, 15×10 5 cells / mL, 10×10 5 cells / mL, 5×10 5 cells / mL, 1.0×10 5 cells / mL, 0.2×10 5 cells / mL.

3. The method for detecting NK cell killing efficiency as described in claim 1, characterized in that, The target cell concentration in the experimental group was 0.5 × 10⁻⁶. 5 cells / mL; The effective-to-target ratios of the experimental groups were 10:1, 50:1, and 100:

1. The NK cell concentrations were 5 × 10⁻⁶. 5 cells / mL, 25×10 5 cells / mL, 50×10 5 cells / mL; The experimental groups were set with effective-to-target ratios of 10:1, 50:1, and 100:

1.

4. The method for detecting NK cell killing efficiency as described in claim 1, characterized in that, In step S5, the formula for calculating the lethality is: Kill rate = [(Experimental group OD1 - Blank group OD1) - (Experimental group OD2 - Blank group OD2)] / (Experimental group OD1 - Blank group OD1) × 100%.

5. The method for detecting NK cell killing efficiency as described in claim 1, characterized in that, It also includes the resuscitation and culture of target cells; the resuscitation and culture of target cells includes the resuscitation and culture of K562 cells and the resuscitation and culture of A549 cells; The resuscitation and culture of the K562 cells include the following steps: S1. Transfer the revived frozen K562 cells to complete culture medium, mix by pipetting, centrifuge and discard the supernatant, then resuspend in complete culture medium and count the cells using an AO / PI cell counter. S2. Add complete culture medium to the cell culture flask, seed K562 cells into the culture flask, and place it in an incubator for static culture. When the cell confluence reaches 80%-90%, pass the cells or freeze them.

6. The method for detecting NK cell killing efficiency as described in claim 5, characterized in that, The resuscitation and culture of A549 cells includes the following steps: S1. Resuscitate the frozen A549 cells and transfer them to complete culture medium. Mix well by pipetting, centrifuge and discard the supernatant. Resuspend the cells in complete culture medium and count the cells using an AO / PI cell counter. S2. Add complete culture medium to the cell culture flask, seed A549 cells into the culture flask, and place it in an incubator for static culture. S3. When the cell confluence reaches 80%-90%, passage or cryopreserve the cells. Remove the culture flask, discard the supernatant, add cell digestive enzymes to digest the cells, observe the cells become rounded, add PBS to stop the digestion, collect the cell suspension, centrifuge and discard the supernatant, resuspend in complete culture medium, and count the cells using an AO / PI cell counter.

7. The kit for detecting NK cell killing efficiency as described in claims 1-6, characterized in that, It includes CCK-8 reagent, target cell culture reagent, and FBS, wherein the target cell culture reagent is selected from RPMI 1640 medium and Ham's F-12K medium.

8. The kit for detecting NK cell killing efficiency as described in claim 7, characterized in that, It also includes a standard curve concentration gradient reference card for establishing a standard curve, the reference card being printed with a target cell concentration gradient of 0.2 × 10⁻⁶. 5 cells / mL up to 1.6 × 10⁻⁶ 5 cells / mL and NK cell concentration gradient 0.2×10 5 cells / mL up to 30 × 10 5 cells / mL.

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