Quantitative detection method for intercellular interaction based on monolayer cell adsorption experiment

Abstract

The invention discloses an intercellular interaction quantitative detection method based on a monolayer cell adsorption experiment, which comprises the following steps: constructing an experiment system conforming to a Langmuir model, and respectively taking cells transfected with different fluorescent label interaction proteins as monolayer recipient cells and ligand cells, after incubation, observing under a fluorescence microscope, counting the number of adsorbed cells, and fitting a corresponding formula to obtain a dissociation constant Kd of the interaction between the cells, so as to realize quantitative evaluation on the affinity. The method provides a new visual angle and tool for studying the interaction between cells and understanding the function exertion of the cells and the interaction of membrane proteins on the surfaces of the cells, and solves the problems that the interaction between the cells cannot be effectively and quantitatively evaluated due to the technical limitation of high requirements on equipment and data, and the membrane proteins are low in content and insoluble in water; and a conventional protein purification method is difficult to obtain a high-purity and high-activity membrane protein sample and is difficult to accurately describe the binding affinity between membrane proteins.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and particularly relates to a method for quantitatively detecting cell interactions mediated by membrane proteins based on a monolayer cell adsorption experiment. Background Art

[0002] Cell-cell interactions (CCIs) are the core mechanism of numerous physiological processes in multicellular organisms and play a crucial role in physiological functions such as tissue formation, nerve conduction, and immune response. The direct physical contact between cells not only drives the formation of tissue and barrier structures but also affects the physiological functions of cells by regulating the changes and activation states of cell signaling pathways. The disruption or abnormality of CCIs may lead to serious pathophysiological consequences.

[0003] CCIs are often driven by membrane proteins. However, the extraction and purification of membrane proteins face many difficulties due to low expression levels and easy loss of native conformation. This results in relatively few studies on their functions despite membrane proteins accounting for 20-30% of all sequenced genomic coding regions. Therefore, in the context of difficult purification and the inability to obtain high-purity proteins with original activity, traditional methods for studying protein-protein interactions are not feasible, and it is easier to conduct in-situ interaction studies of proteins on the cell membrane.

[0004] In recent years, with the continuous development of biotechnology, a variety of experimental methods have been used to observe and track cell-cell interactions. The development of optical microscopy technology has enabled researchers to directly observe the cell-cell contact interface and understand the spatial and organizational structures of these interactions. For example, through the supported planar lipid bilayer technique, researchers can track the movement and organization of ligands-receptors on the cell membrane during cell-cell interactions; the application of methods such as super-resolution imaging technology and fluorescence protein complementation strategies (such as GFP reconstitution imaging technology) has further improved the visualization and tracking ability of cell-cell interactions; fluorescence correlation spectroscopy can focus on the fluorescence fluctuations at the cell contact surface to analyze the molecular diffusion coefficient and interactions.

[0005] In addition to imaging techniques, chemical labeling methods also provide a new perspective for studying cell-cell interactions. Contact-dependent labeling techniques (such as LIPSTIC and EXCELL) and non-contact-dependent labeling techniques (such as APEX and BioID) can identify directly interacting cells and biomolecules at the cell-cell interface by activating labeling probes in the cell-cell contact environment. Additionally, to conduct a simpler and more effective quantitative exploration and visualization of CCIs, researchers have developed methods such as cell aggregation experiments and cell-cell contact experiments to reveal the characteristics of cell-cell interactions by observing the aggregation and contact of cells under specific conditions.

[0006] However, most of the above methods can only provide qualitative or semi - quantitative information, and it is difficult to uniformly and accurately describe the kinetic characteristics of cell - cell interactions. In addition, the existing methods also have certain limitations in experimental design and data analysis, such as the complexity of experimental equipment and required techniques, and the difficulty in monitoring the dynamic changes of cell - cell interactions. Summary of the Invention

[0007] The purpose of the present invention is to provide a quantitative detection method for cell - cell interactions based on monolayer cell adsorption experiments to solve the problem that it is difficult to uniformly and accurately describe the kinetic characteristics of cell - cell interactions in the prior art.

[0008] To achieve the above - mentioned purpose, the present invention adopts the following technical solutions:

[0009] A quantitative detection method for cell - cell interactions based on monolayer cell adsorption experiments, comprising the following steps:

[0010] Step 1, cell transfection: Construct an experimental system that conforms to the Langmuir model. Cells transfected with different fluorescent - tagged interacting proteins are used as monolayer receptor cells (stationary phase) and ligand cells (mobile phase) respectively. The binding between the two types of cells is 1:1 and the binding events are independent of each other;

[0011] Step 2, cell collection and counting: After transfection, the ligand cells are collected with dissociation buffer, the supernatant is discarded after centrifugation, and the cells are resuspended in incubation buffer to form a single - cell suspension and counted;

[0012] Step 3, cell incubation: Remove the medium in the receptor cells, add ligand cells to the corresponding well plates according to the cell - number gradient, and make up with incubation buffer. Incubate the system on a shaker, then discard the cell - suspension medium, and wash the cells with washing buffer multiple times to remove non - specifically adsorbed cells;

[0013] Step 4, image acquisition: Use a fluorescence inverted microscope for image acquisition;

[0014] Step 5, data analysis: Use ImageJ to count the number of ligand cells in the photographed field of view;

[0015] Step 6, formula fitting: Import the counted data into the saturation - curve formula for fitting to obtain the dissociation constant K of the cells d , realizing the quantitative evaluation of the affinity.

[0016] Preferably, in step 1, the cells are seeded in a 24 - well plate for 24 h. After the cell confluence reaches 60% - 70%, plasmids expressing target molecule A and target molecule B are transfected into receptor cells and ligand cells respectively.

[0017] Preferably, target molecule A and target molecule B are any two membrane protein molecules that interact with each other.

[0018] Preferably, the plasmid expressing target molecule A and the plasmid expressing target molecule B each carry different fluorescent protein tags.

[0019] Preferably, in step 1, when transfecting cells, an empty plasmid carrying the same fluorescent group as the target molecule plasmid needs to be co-transfected, and the transfection ratio is target molecule plasmid: empty plasmid = 3:1 (w / w).

[0020] Preferably, in step 2, 48 hours after transfection, the paired cells are collected with dissociation buffer.

[0021] Preferably, in step 2, the dissociation buffer is phosphate buffer with a final concentration of 1 mM ethylenediaminetetraacetic acid (EDTA).

[0022] Preferably, in step 2, the incubation buffer is based on DMEM medium, containing 50 mM HEPES-NaOH with a pH of 7.4, 10% fetal bovine serum (FBS) by volume fraction, 10 mM CaCl2, and 10 mM MgCl2 at final concentrations.

[0023] Preferably, in step 3, the cell number gradient range is greater than two orders of magnitude.

[0024] Preferably, in step 3, the volume is made up to 500 μL with incubation buffer.

[0025] Preferably, in step 3, the system is incubated on a shaker at 37°C / 25°C at 90 rpm.

[0026] Preferably, in step 3, the incubation time is the time required for the bound paired cells to reach saturation. By fixing the concentration of paired cells in kinetic experiments and measuring the binding ability of paired cells to receptor cells at different time points, the optimal incubation time is determined.

[0027] Preferably, in kinetic experiments, the concentration of paired cells is fixed, and the binding of paired cells at each time point of 15 min, 30 min, 45 min, 60 min, 75 min, and 90 min of incubation is measured. It is determined that the bound paired cells tend to saturate after 60 min of incubation, and the optimal incubation time is 60 min.

[0028] Preferably, in step 3, the washing buffer is phosphate buffer containing 0.01% Tween-20 by final volume concentration.

[0029] Preferably, in step 4, image acquisition is performed under a 10X objective lens.

[0030] Preferably, in step 6, the saturation curve formula includes two cases: considering ligand consumption or not considering ligand consumption. The derivation is as follows:

[0031] (1) Definition: In the monolayer cell adsorption experiment, the ligand cells are defined as L, and their cell density is [L]; the receptor cells available for binding are defined as R, and their cell density is [R]; the receptor-ligand cell complex is defined as RL, that is, the ligand cells that have bound to the receptor cells, and its concentration is [RL]. When the binding between the two reaches equilibrium:

[0032]

[0033] At this time, R][L]k on =[RL]k off , k on is the binding rate constant, with the unit of concentration -1 time -1 , k off is the dissociation rate constant, with the unit of time -1 ; the dissociation constant K d is defined as:

[0034]

[0035] The dissociation constant K d has the unit of concentration;

[0036] (2) When not considering ligand consumption:

[0037] The saturation experiment is to measure the binding ability when the binding between the ligand and the receptor reaches equilibrium at a series of ligand concentrations over a long enough time, so as to calculate the dissociation constant. Without considering ligand consumption, that is, the concentration of the ligand cells is considered to remain constant during the experiment and always equal to the initial value. At this time, the concentration relationship between the ligand cells and the receptor cells at time t is as follows:

[0038] [L total =[L t ;

[0039] [R total =[R t +[RL t

[0040] Among them, [L total is the total concentration of the added ligand cells, [L t is the concentration of the free ligand cells at time t, [R total is the total concentration of the added receptor cells, [R t is the concentration of the remaining receptor cells available for binding at time t, [RL t ​is the concentration of the donor-recipient cell complex at time t;

[0041] Therefore, Equation (1) is transformed into:

[0042]

[0043] When the binding between the two reaches equilibrium, [RL t is a constant value, [RL t = [RL max ; where [RL max is the theoretical maximum concentration of the donor-recipient cell complex; in the monolayer cell adsorption experiment, if the time is fixed and made long enough to reach the plateau phase, and the concentration of the ligand cells added [L total is set as the independent variable, a scatter plot of [RL t - [L total can be obtained, and thus the dissociation constant K d can be obtained by fitting;

[0044] By using the reciprocal plotting method (Scatchard Plot) for Equation (1), we get:

[0045]

[0046] The ordinate is The abscissa is plotted with [RL t , and the obtained scatter plot can be fitted into a straight line with a negative slope, which is used to evaluate the reliability of the experimental system;

[0047] (3) Considering ligand consumption: When the ligand cells bind to the receptor cells, the concentration of free ligand cells will decrease, and this process is ligand consumption. The ligand consumption coefficient δ is defined as:

[0048]

[0049] When 50% of the receptor binding sites are occupied by the ligand cells, according to Equation (2), at this time, the concentration of free ligand [L t = K d , and the total ligand concentration added under this condition is defined as EC 50 ; Therefore:

[0050]

[0051] At the ligand concentration of EC 50 :

[0052]

[0053] According to Equation (2), if ligand consumption is not considered, that is, [L total = [Lt , EC 50 is numerically equal to K d value; when [R total < 0.1K d , the ligand consumption < 5%, and at this time EC 50 and K d value has an error within 5%, and the existence of ligand consumption can be ignored; if not this case, the ligand consumption needs to be included in the mathematical model consideration range, and at this time:

[0054] [L total = [L t + [RL t ;

[0055] [R total = [R t + [RL t

[0056] The process of ligand cells and receptor cells binding can be described as:

[0057]

[0058] In the saturation experiment, the incubation time of ligand cells and receptor cells is long enough, and the binding-dissociation process has reached dynamic equilibrium, that is:

[0059]

[0060] [RL t 2 - [RL t [R total + [L total + K d ) + [R total [L total

[0061] In this formula, the status of ligand and receptor cells is indistinguishable, and the solution is:

[0062]

[0063] Equation (8) is the fitting formula for the saturation experiment considering ligand consumption in the single-layer cell adsorption experiment, and the dissociation constant K d .

[0064] The present invention constructs an experimental system that conforms to the Langmuir model, uses the interacting cells as the receptor stationary phase (R) and the ligand free phase (L) respectively, their binding is 1:1 and the binding events are independent of each other, counts the adsorption quantity of the free phase cells and fits the corresponding formula to obtain the dissociation constant K d . K​​​d The unit is concentration, representing the dissociation degree of RL at equilibrium. K d The larger the value, the more dissociation occurs and the weaker the affinity between RL; conversely, the smaller K d The smaller the value, the less dissociation occurs and the stronger the affinity between RL. Its physical meaning is that when the ligand concentration is equal to K d half of the receptors are bound to the ligand.

[0065] Among them, the kinetic experiment is to fix the concentration of ligand cells and measure the binding ability of ligand cells and receptor cells at different time points to determine the incubation time of the saturation experiment.

[0066] The saturation experiment is to measure the results when the binding between the ligand and the receptor reaches equilibrium at a series of ligand cell concentrations with a long enough incubation time, so as to calculate the dissociation constant K d .

[0067] When ligand consumption is not considered, that is, the concentration of ligand cells is considered to remain constant during the experiment and always equal to the initial concentration, it conforms to Equation (2) at equilibrium.

[0068] When ligand consumption is considered, that is, when ligand cells and receptor cells bind, the concentration of free ligand cells will decrease, and at this time it conforms to Equation (8) at equilibrium.

[0069] The present invention requires observing and analyzing cells, which is achieved by co-transfecting a recombinant plasmid carrying a fluorescent tag and an empty plasmid carrying only the fluorescent tag.

[0070] In the present invention, the counting of adsorbed cells is achieved by obtaining images under a fluorescence microscope and using the cell counting function of ImageJ, and the formula fitting is completed in Origin software.

[0071] The present invention applies the monolayer cell adsorption experiment method to the cell interaction based on the binding of Nrxn1α / βSS4- and LRRTM2, and explores the effects of temperature and ligand cell consumption on the K d value.

[0072] The present invention applies the monolayer cell adsorption experiment method to the cell interaction based on the binding of Nrxn1α / βSS4- and CL1, and explores the effects of non-specific adsorption on the K d value.

[0073] The present invention applies the monolayer cell adsorption experiment method to the cell interaction based on the binding of ICAM-1 and LFA-1, and explores the effects of the expression abundance of membrane proteins on the K d value.

[0074] The content of the present invention also includes obtaining the theoretical maximum receptor cell concentration [R that reflects the available ligand cell binding through the monolayer cell adsorption experimentT or [RL max , which is relatively independent of the K d value and represents different aspects of cell binding ability.

[0075] Beneficial effects: The present invention designs a strategy called monolayer cell adsorption experiment, fits the experimental results with kinetic formulas, and can obtain the K d and [R T calculated value. The present invention applies this experimental strategy to the cell binding induced by the interaction between Nrxn1α / βSS4- and LRRTM2, CL1 in nerve synapses and the interaction between LFA-1 and ICAM-1 in immune synapses, realizing the quantitative detection of cell binding between different types of cells. And it has confirmed the influence of various factors such as temperature, ligand consumption, protein interaction type, non-specific adsorption and membrane protein expression abundance on the determination of cell binding ability. The present invention develops a simple and effective method for quantitatively evaluating cell-cell interactions, which helps to reveal the molecular mechanism of cell communication and the network regulation logic, makes up for the blank of research methods in this aspect to a certain extent, and shows great application potential. Brief Description of the Drawings

[0076] Figure 1 It is a schematic diagram and process of monolayer cell adsorption experiment.

[0077] Figure 2 It is the saturation experiment at different temperatures in Example 1 (taking LRRTM2 and Nrxn1αSS4- as an example); where: (a) Cell adsorption at different temperatures and time points with a fixed number of free ligand cells; (b) Statistical chart; [L total = 2.0*10^5, Temp = 25°C, 10x, Scalebar = 100μm, n = 3.

[0078] Figure 3 It is the cell counting and formula fitting of monolayer cell adsorption experiment based on the interaction between LRRTM2 and Nrxn1αSS4- in Example 2; where: (a) The independent variable is the initial number of ligand cells, and the dependent variable is the number of adsorbed cells in each field of view; (b) Conversion of the total number of adsorbed cells and cell number to concentration; (c) Formula fitting, the plotting unit is the cell number, without considering ligand consumption; (d) The plotting unit is the cell number, considering ligand consumption; (e) The plotting unit is the cell concentration, considering ligand consumption. 37°C, n = 3.

[0079] Figure 4Cell counting and formula fitting for the monolayer cell adsorption experiment based on the interaction between LRRTM2 and Nrxn1αSS4 in Example 3; where: (a) The independent variable is the initial ligand cell number, and the dependent variable is the adsorbed cell number per field of view; (b) Conversion of the total adsorbed cell number and cell number to concentration; (c) Formula fitting, with the plotting unit being cell number and ligand consumption not considered; (d) Formula fitting, with the plotting unit being cell number and ligand consumption considered; (e) Formula fitting, with the plotting unit being cell concentration and ligand consumption considered. 25 °C, n = 3.

[0080] Figure 5 Data processing and formula fitting for the monolayer cell adsorption experiment based on the interaction between LRRTM2 and Nrxn1βSS4 in Example 4; where: (a) The independent variable is the initial ligand cell number, and the dependent variable is the adsorbed cell number per field of view; (b) Conversion of the total adsorbed cell number and cell number to concentration; (c) Formula fitting, with the plotting unit being cell number and ligand consumption not considered; (d) Formula fitting, with the plotting unit being cell number and ligand consumption considered; (e) Formula fitting, with the plotting unit being cell concentration and ligand consumption considered. 37 °C, n = 3.

[0081] Figure 6 Monolayer cell adsorption experiment based on the interaction between CIRL1 / latrophilin-1 (CL1) and Nrxn1α / βSS4 in Example 5; where: (a) Results of the monolayer cell adsorption experiment, taking the ligand cell number as 1.5*10^6 as an example; 10x, Scale bar = 100 μm; (b) Statistics of the number of ligand cells bound per field of view; (c) Net value of cell-cell binding caused by membrane protein interaction per field of view after removing non-specific adsorption, taking CL1 and Nrxn1αSS4- as an example. n = 6.

[0082] Figure 7 Data processing and fitting in Example 5; where: (a)-(c): CL1 + Nrxn1αSS4-; (d)-(f): CL1 + Nrxn1βSS4-; (a)&(d): Plotting with the total cell number and cell concentration as units respectively after removing non-specific adsorption; (b)&(e): Retaining the mean value to make a scatter plot and performing curve fitting to obtain kinetic parameters (ligand compensation not considered); (c)&(f): Retaining the mean value to make a scatter plot and performing curve fitting to obtain kinetic parameters (ligand compensation considered). n = 6.

[0083] Figure 8 Scatchard plots in Example 5; where: (a) CL1 + Nrxn1αSS4- CCIs, Adj R-square = 0.82; (b) CL1 + Nrxn1βSS4- CCIs, Adj R-square = 0.57.

[0084] Figure 9 It is the monolayer cell adsorption experiment based on the interaction between Itgal1 / Itgb2 (LFA-1) and ICAM-1 in Example 6; wherein: (a) The results of the monolayer cell adsorption experiment, taking the number of ligand cells as 1.0×10^6 as an example; 10x, Scale bar = 100 μm; (b) The number of bound cells in each field of view; (c) Plotting graphs with the total number of cells and cell concentration as units respectively; (d) Making a scatter plot with the average value retained in terms of the number of cells and performing formula fitting; (e) Making a scatter plot with the average value retained in terms of cell concentration and performing formula fitting. n = 9.

[0085] Figure 10 It is the influence of the expression abundance of membrane proteins on the cell-cell binding ability in Example 6; wherein: (a) The plasmid transfection protocol for adherent monolayer cells in the stationary phase, pEGFP-C1:pICAM-1-EGFP = 3:1 or pEGFP-C1:pICAM-1-EGFP = 1:3; (b) Performing Western Blot analysis on the expression of the target protein in the cells transfected with the two protocols; (c) Performing semi-quantitative analysis on the results in Figure (b) and calculating the expression level of the target protein in each group compared to the internal reference protein; n = 3; (d) Performing statistical analysis on the results of the monolayer cell adsorption experiment carried out according to the two transfection protocols, and plotting graphs with the total number of cells and cell concentration as units respectively; (e) Making a scatter plot with the average value retained in terms of the number of cells and performing formula fitting; (f) Making a scatter plot with the average value retained in terms of cell concentration and performing formula fitting. n = 9. Detailed implementation manners

[0086] The present invention will be further explained below in conjunction with examples and the accompanying drawings.

[0087] Example 1

[0088] Saturation experiment (at 25°C and 37°C) of cell interaction based on the binding of LRRTM2 and Nrxn1αSS4-.

[0089] Cell transfection: For easy observation, when performing cell transfection, an empty plasmid carrying the same fluorescent group as the target molecule plasmid needs to be co-transfected. The cells are seeded in a 24-well plate for 24 h. After the cell confluence reaches 60% - 70%, the plasmids expressing the target molecule LRRTM2 (pmCherry-LRRTM2 + pmCherry-N1, 3:1, w / w) and the target molecule Nrxn1αSS4- (pEGFP-Nrxn1αSS4- + pEGFP-C1, 3:1, w / w) are transfected into donor cells and recipient cells respectively.

[0090] Cell collection and counting: To ensure the validity of the formula for explaining this experimental model, the transfected cells in the 24-well plate had a confluence of more than 95% and were evenly distributed 48 hours after transfection. The donor cells were collected with dissociation buffer (phosphate buffer with a final concentration of 1 mM EDTA), and the supernatant was discarded after centrifugation. The cells were resuspended in incubation buffer: DMEM (WISENT, CAT.NO.319-005-CL) containing 50 mM HEPES-NaOH, pH = 7.4, 10% FBS (Excell, FSS500), 10 mM CaCl2, and 10 mM MgCl2 to form a single-cell suspension, and then counted using a hemocytometer.

[0091] Cell incubation: Discard the medium in the 24-well plate of recipient cells. Add the ligand cells to the corresponding wells according to the set cell number gradient, and make up the volume to 500 μL with incubation buffer. Set three replicates for each group. Incubate the system on a shaker at 37 °C or 25 °C at 90 rpm for a certain period of time (set time gradient). Then discard the cell suspension medium, and wash the cells four times with phosphate buffer containing 0.01% Tween-20 to remove non-specifically adsorbed cells.

[0092] Image acquisition: Use a Leica inverted fluorescence microscope to acquire images under a 10x objective lens, and capture images in two channels respectively.

[0093] Data analysis: Use ImageJ for batch cell counting, and only count the ligand cells in the field of view, that is, the number of mCherry fluorescent cells. The specific steps are as follows: First, randomly open a representative image, click "Plugins - Macros - Record" to record a single operation, then click "Image - Type - 8bit" -> "Edit - Invert" -> "Imgae - Adjust - Threshold" -> "Analyze - Binary - Fill Holes - Watersheld" -> "Analyze - Set Scale" -> "Analyze - Analyze Particles" to obtain the number of cells in the field of view and generate Macro code. Then click "Process - Batch - Macro" in sequence for batch cell counting.

[0094] It was found that the cell adhesion ability based on the binding of LRRTM2 and Nrxn1αSS4 - at 37 °C was stronger than that at 25 °C.

[0095] Example 2

[0096] Kinetic experiment on cell interaction based on the binding of LRRTM2 and Nrxn1αSS4 - (37 °C).

[0097] Cell transfection, cell collection and counting, cell incubation, image acquisition, and data analysis were the same as in Example 1, except that the fixation time during cell incubation was 60 min.

[0098] Formula fitting: For each group of cells, the counting data was imported into Origin. Equations (2) and (8) were newly created in "Tools - Fitting Function Builder" of Origin. A scatter plot was drawn based on the cell counting results, and then the user-defined formula was found in "Analyze - Fitting - Nonlinear Curve Fit". After defining the initial parameter values, fitting was performed to obtain the dissociation constant K between cells d , the maximum binding cell density [R T (or RL max ) and the fitting effect information. Using Equation (2) for fitting, K d,obs = (3.01 ± 0.57) × 10 5 , with the unit of cell number, without considering ligand consumption; when ligand consumption was taken into account and Equation (8) was used for fitting, K d,obs = (9.87 ± 2.56) × 10 4 , and converting to concentration units, K d = 0.328 ± 0.085 fM.

[0099] Example 3

[0100] Kinetic experiment on cell interaction based on the binding of LRRTM2 to Nrxn1αSS4- (25 °C).

[0101] The implementation method was the same as in Example 2, except that the incubation time was changed to 25 °C.

[0102] Using Equation (2) for fitting, K d,obs = (3.98 ± 0.48) × 10 5 , with the unit of cell number, without considering ligand consumption; when ligand consumption was taken into account and Equation (8) was used for fitting, K d,obs = (2.55 ± 0.30) × 10 4 , and converting to concentration units, K d = 0.848 ± 0.099 fM.

[0103] Example 4

[0104] Kinetic experiment on cell interaction based on the binding of LRRTM2 to Nrxn1βSS4- (37 °C).

[0105] The implementation method is the same as that of Example 2, except that the plasmid transfected into the recipient cells is changed to pEGFP-Nrxn1βSS4- + pEGFP-C1, 3:1, w / w.

[0106] K was obtained by fitting with Equation (2) d,obs =(1.26±0.28)×10 5 , with the unit of cell number, without considering ligand consumption; when ligand consumption was taken into account and fitted with Equation (8), K d,obs =(8.15±0.27)×10 4 , and when converted to the concentration unit, K d =0.271±0.090 fM.

[0107] Example 5

[0108] Kinetic experiment (37 °C) on cell interaction based on the binding of CL1 to Nrxn1α / βSS4-.

[0109] The implementation method is the same as that of Example 2, except that the plasmid transfected into the donor cells is changed to pmCherry-CL1 + pmCherry-N1, 3:1, w / w; the plasmid transfected into the recipient cells is changed to pEGFP-Nrxn1α / βSS4- + pEGFP-C1, 3:1, w / w. When performing cell counting, the statistical values of each point in the experimental group were subtracted by the number of non-specifically adsorbed cells in the control group to obtain the net number of adsorbed cells. After removing non-specific adsorption and fitting with Equation (2), the K of cell interaction based on the binding of CL1 to Nrxn1αSS4- was obtained as d =6.51±1.19 fM, and the K based on the binding of CL1 to Nrxn1βSS4- was d =6.13±1.42 fM; when ligand consumption was taken into account and fitted with Equation (8), the K d values were 6.40±1.17 fM and 5.98±1.38 fM, respectively.

[0110] Scatchard analysis and plotting were performed using Equation (3), and it was found that although the distribution of recipient cells in this experimental system was not completely uniform, within a certain range, the degree of surface site heterogeneity was not sufficient to determine the presence of cooperative effects or other complex mechanisms.

[0111] Example 6

[0112] Kinetic experiment (37 °C) on cell interaction based on the binding of LFA-1 to ICAM-1.

[0113] The implementation method is the same as that of Example 2, except that the plasmid transfected into the donor cells is pmCherry-LFA-1 + pmCherry-N1, 3:1, w / w; the plasmid transfected into the recipient cells is pEGFP-ICAM-1 + pEGFP-C1, 3:1 or 1:3, w / w.

[0114] When fitting with formula (8), the relative abundance of ICAM-1 expression in the group with 1.466 ± 0.077 was K d = 1.93 ± 0.39 fM; the relative abundance of ICAM-1 expression in the group with 1.004 ± 0.037 was K d = 2.66 ± 1.06 fM, and ligand consumption was taken into account in both cases.

[0115] The above are only the preferred implementation modes of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A method for quantitatively detecting cell - cell interactions based on a single - layer cell adsorption experiment, characterized in that: The following steps are involved: Step 1, cell transfection: construct an experimental system that conforms to the Langmuir model, and use cells transfected with different fluorescent-tagged interacting proteins as monolayer receptor cells and ligand cells, respectively. The binding between the two cells is 1:1 and the binding events are independent of each other; Step 2, cell collection and counting: After transfection, the ligand cells were collected with dissociation buffer, centrifuged and the supernatant was discarded, resuspended with incubation buffer to a single cell suspension, and counted; Step 3, cell incubation: remove the culture medium in the receptor cells, add ligand cells to the corresponding well plate according to the cell number gradient, and fill with incubation buffer, place the system on a shaker for incubation, then discard the cell suspension culture medium, rinse the cells with washing buffer several times to remove non-specific adsorbed cells; Step 4, image acquisition: using a fluorescence inverted microscope to acquire images; Step 5, data analysis: Use ImageJ to count the number of ligand cells in the field of view; Step 6, formula fitting: Import the count data into the saturation curve formula for fitting to obtain the dissociation constant K between cells d , achieving quantitative evaluation of the affinity.

2. The method for quantitatively detecting cell-cell interaction based on a single-layer cell adsorption experiment according to claim 1, wherein: In step 1, cells are seeded into a 24-well plate for 24 hours. After the cell confluence reaches 60% to 70%, the plasmids expressing target molecule A and target molecule B are transfected into receptor cells and ligand cells respectively.

3. The method for quantitatively detecting cell-cell interaction based on monolayer cell adsorption experiment according to claim 2, wherein: Target molecule A and target molecule B are any two interacting membrane protein molecules.

4. The method for quantitatively detecting cell-cell interactions based on a single-layer cell adsorption experiment according to claim 2, characterized in that: The plasmid expressing the target molecule A and the plasmid expressing the target molecule B each carry a different fluorescent protein tag.

5. The method for quantitatively detecting cell-cell interaction based on monolayer cell adsorption experiment according to claim 1, wherein: In step 1, when cells are transfected, an empty plasmid carrying the same fluorescent group as the target molecule plasmid needs to be co-transfected, and the transfection ratio is target molecule plasmid: empty plasmid = 3:1 (w / w).

6. The method for quantitatively detecting cell-cell interaction based on monolayer cell adsorption experiment according to claim 1, wherein: In step 2, the dissociation buffer is a phosphate buffer with a final concentration of 1 mM ethylenediaminetetraacetic acid (EDTA); The incubation buffer is based on DMEM culture medium, containing 50 mM hydroxyethylpiperazine ethanesulfonic acid (HEPES)-NaOH at a final concentration of pH-7.4, 10% fetal bovine serum (FBS) by volume, 10 mM CaCl2, and 10 mM MgCl2.

7. The method for quantitatively detecting cell-cell interaction based on monolayer cell adsorption experiment according to claim 1, wherein: In step 3, the range of the cell number gradient is greater than two orders of magnitude.

8. The method for quantitatively detecting cell-cell interaction based on monolayer cell adsorption experiment according to claim 1, wherein: In step 3, the incubation time is the time required for the bound ligand cells to reach saturation. The ligand cell concentration is fixed by kinetic experiments, and the binding ability of the ligand cells to the receptor cells at different time points is measured to determine the optimal incubation time; In the kinetic experiment, the ligand cell concentration was fixed, and the binding of ligand cells at each time point was measured at 15 min, 30 min, 45 min, 60 min, 75 min, and 90 min of incubation. It was determined that the bound ligand cells approached saturation after 60 min of incubation, and the optimal incubation time was 60 min.

9. The method for quantitatively detecting cell-cell interaction based on monolayer cell adsorption experiment according to claim 1, characterized in that: In step 3, the washing buffer is a phosphate buffer containing Tween-20 at a final volume concentration of 0.01%.

10. The method for quantitatively detecting cell-cell interaction based on monolayer cell adsorption experiment according to claim 1, wherein: In step 6, the saturation curve formula includes two cases: considering ligand consumption or not considering ligand consumption, and is derived as follows: (1) Definition: In the single-layer cell adsorption experiment, the ligand cells are defined as L, and their cell concentration is [L]; the receptor cells available for binding are defined as R, and their cell concentration is [R]; the ligand-receptor cell complex is defined as RL, that is, the ligand cells that have bound to the receptor cells, and its concentration is [RL]; when the binding between the two reaches equilibrium: At this time, [R][L] kon = [RL]k off , k on is the association rate constant, with the unit of concentration -1 time -1 , k off is the dissociation rate constant, with the unit of time -1 ; The dissociation constant K d is defined as: Dissociation constant K d Unit: concentration; (2) When ligand consumption is not considered: The saturation experiment is to measure the binding ability when the binding between the ligand and the receptor reaches equilibrium at a series of ligand concentrations over a sufficiently long time, so as to calculate the dissociation constant; without considering ligand consumption, that is, the concentration of ligand cells is considered to remain constant during the experiment and always equal to the initial value. At this time, the concentration relationship between ligand cells and receptor cells at time t is as follows: [L total =[L t ; [R total = [R t + [RL t ​ where, [L total is the total concentration of added ligand cells, [L t is the concentration of free ligand cells at time t, [R total is the total concentration of added receptor cells, [R t is the concentration of remaining receptor cells available for binding at time t, [RL t is the concentration of ligand-receptor cell complexes at time t; Therefore, Equation (1) is transformed into: When the combination between the two reaches equilibrium, [RL t is a constant value, [RL t = [RL max , where [RL max is the theoretical maximum concentration of the donor - recipient cell complex; in the monolayer cell adsorption experiment, if the time is fixed and made long enough to reach the plateau phase, setting the concentration of the ligand - added cells [L total as the independent variable, a scatter plot of [RL t - [L total can be obtained, and thus the dissociation constant K d can be fitted; If the reciprocal plotting method (Scatchard Plot) is performed on Equation (1), we get: The vertical coordinate is The horizontal coordinate is [RL t . By plotting, the obtained scatter plot can be fitted to a straight line with a negative slope, which is used to evaluate the reliability of the experimental system; (3) When ligand consumption is considered: When ligand cells bind to receptor cells, the concentration of free ligand cells will decrease. This process is ligand consumption, and the ligand consumption coefficient δ is defined as: When 50% of the receptor binding sites are occupied by the ligand cells, according to Equation (2), the concentration of the free ligand [L t = K d , and the total ligand concentration added under this condition is defined as EC 50 ; therefore: At a ligand concentration of EC 50 : As can be seen from Equation (2), if ligand consumption is not considered, i.e., [L total = [L t , EC 50 is numerically equal to the K d value; when [R total < 0.1K d , the ligand consumption < 5%, and at this time the error between EC 50 and the K d value is within 5%, so the existence of ligand consumption can be ignored; if not in this case, the ligand consumption needs to be included in the scope of consideration of the mathematical model. At this time: [L total =[L t +[RL t ; [R total = [R t + [RL t ​ The binding process between ligand cells and receptor cells can be described as: In the saturation experiment, the incubation time of ligand cells and receptor cells is long enough, and the binding-dissociation process has reached dynamic equilibrium, that is: [RL t 2 -[RL t ([R total +[L total +K d )+[R total [L total ​​ In this formula, the ligand and receptor cells have no difference in status, and the solution is: Equation (8) is the fitting formula for the saturation experiment considering ligand consumption in the single-layer cell adsorption experiment, and thus the dissociation constant K can be obtained. d .

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