CN115980356A - Method, kit and use for detecting epithelial-mesenchymal transition of cells - Google Patents

Abstract

The invention provides a method, a kit and application for detecting epithelial-mesenchymal transition of cells, wherein the method comprises the following steps: 1) Incubating the integrin antibody with single biotin with the cells to be detected to specifically recognize and bind integrin on the cell membrane surface of the cells, and removing unbound integrin antibody with single biotin after the incubation is completed; 2) Adding a fluorescent probe with streptavidin into the obtained substance in the step 1), incubating, and removing the unreacted redundant fluorescent probe with streptavidin after reaction to obtain the integrin marked by the fluorescent probe; 3) Detecting a change in the average diffusion rate of the fluorescent probe-labeled integrin relative to control cells by imaging the fluorescent probe for the fluorescent probe-labeled integrin obtained in step 2), wherein an increase in the average diffusion rate of the fluorescent probe-labeled integrin relative to control cells indicates epithelial-to-mesenchymal transition of the cells. The invention has higher sensitivity and accuracy and can effectively reduce the false negative rate in measurement.

Description

Method, kit and use for detecting epithelial-mesenchymal transition of cells

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for detecting epithelial-mesenchymal transition of cells.

Background

Epithelial-mesenchymal transition (EMT) refers to the transition of epithelial cells to mesenchymal cells, is an important biological process, and has important significance for the study of cell differentiation, embryonic development, cancer metastasis, tissue healing, and the like. After EMT, the cells obtain strong invasive ability, and the moving speed of the cells is improved. The current common EMT is marked by: 1. the cell shape change is changed into fusiform by fish scale deformation; 2. reduction of cadherin (E-cadherin), a marker of cell-to-cell junctions; 3. cytoskeleton changes, and obvious sheet pseudopodia appears; 4. has abundant intermediate filaments. The common way of determining EMT is therefore mainly to measure changes in cell morphology or changes in cadherin. However, these two methods do not perfectly distinguish the cells after EMT induction.

During cell migration, focal Adhesion (FAS), the link between cells and the extracellular matrix (ECM), plays an important role. The adhesive spots are dynamic molecular polymerization and are continuously decomposed and polymerized. Where integrins are an important part of the composition of focal adhesions, focal adhesion formation can be controlled by modulating the affinity of the integrins for the ECM. Integrin is a transmembrane protein, having two states, activated and inactivated, and switching between the two states. When it has not been activated, it moves across the cell membrane. When activated, it recruits proteins such as Talin to link the extracellular matrix to actin, forming contractile units. After activation, integrin is in a state of temporary fixation in which one end is linked to the outside and one end is linked to actin. The complex formed by multiple integrins matures to form Focal Adhesion (FAS). The physical connection at the focal adhesion pulls the cells forward during cell movement. Integrins are therefore important proteins closely related to cellular motility.

A series of studies have been conducted on integrin labeling, integrin motility, including different movement states of integrin inside and outside focal adhesions, integrin halting and activation, integrin diffusion behavior, and the like. Current single molecule studies on integrins focus mostly on the problem of FAS formation and integrin activation. In addition, there are some studies that continue in vivo as integrin single molecule studies in vitro, involving how integrin on a small scale participates in the behavior of focal adhesion, rather than the large scale problem of cell crawling. Currently, the studies on integrin and EMT are limited to biological signaling pathway related studies, and there is no study on integrin kinetics in combination with EMT.

Disclosure of Invention

It is an object of the present invention to provide a novel method for detecting epithelial-to-mesenchymal transition of cells. Unlike previous methods of labeling integrin by ligand-Quantum Dot (QD)/GFP binding, the present inventors have unexpectedly discovered that labeling integrin by binding a primary antibody with a single biotin to a fluorescent probe with streptavidin allows differentiation between E-type cells and M-type cells to be demonstrated by diffusion movement of the integrin, thereby enabling detection of epithelial-mesenchymal transition of cells. Based on this finding, the present invention has been completed.

It is another object of the present invention to provide a kit for detecting epithelial-to-mesenchymal transition of cells.

It is a further object of the invention to provide the use of said kit.

The purpose of the invention is realized by the following technical scheme:

in one aspect, the invention provides a method of detecting epithelial-to-mesenchymal transition of a cell, the method comprising the steps of:

1) Incubating the integrin antibody with single biotin with the cells to be detected to specifically recognize and bind integrin on the cell membrane surface of the cells, and removing unbound integrin antibody with single biotin after the incubation is completed;

2) Adding a fluorescent probe with streptavidin into the obtained substance in the step 1), incubating, and removing the unreacted redundant fluorescent probe with streptavidin after reaction to obtain the integrin marked by the fluorescent probe;

3) Detecting a change in the average diffusion rate of the fluorescent probe-labeled integrin relative to control cells by imaging the fluorescent probe for the fluorescent probe-labeled integrin obtained in step 2), wherein an increase in the average diffusion rate of the fluorescent probe-labeled integrin relative to control cells indicates epithelial-to-mesenchymal transition of the cells.

Preferably, the average diffusion rate of the fluorescent probe-labeled integrin is increased by 40% to 172% relative to control cells; more preferably, the average diffusion rate of the fluorescent probe-labeled integrin is increased by 80% to 172% relative to control cells.

Preferably, a step of pretreating the cells is further included before the step 1).

Preferably, the cells are selected from one or more of epithelial cells, stromal cells, tumor cells, peripheral blood mononuclear cells or lymphocytes; more preferably, the cells are epithelial cells or mesenchymal cells; most preferably, the cell is a normal breast cell MCF10A or an epithelial-mesenchymal transformed breast cell MCF10A.

Preferably, the control cells are selected from the group consisting of corresponding cells that have not undergone epithelial-to-mesenchymal transition; more preferably, the control cell is an epithelial cell; most preferably, the control cell is a normal breast cell MCF10A.

Preferably, in step 1), the incubation time is 10 to 18 minutes and the incubation temperature is 0 to 4 ℃; more preferably, the incubation time is 15 minutes and the incubation temperature is 0 ℃.

Preferably, the integrin antibody with single biotin is selected from one or more of an anti- β 1 integrin antibody with single biotin, an anti- α 5 integrin antibody with single biotin, an anti- β 3 integrin antibody with single biotin, an anti- α v integrin antibody with single biotin; more preferably, the integrin antibody with a single biotin is an anti- β 1 integrin antibody or an anti- α 5 integrin antibody with a single biotin; most preferably, the integrin antibody with a single biotin is an anti- β 1 integrin antibody with a single biotin.

Preferably, in step 2), the incubation time is 3 to 8 minutes and the incubation temperature is 0 to 4 ℃; more preferably, the incubation time is 5 minutes and the incubation temperature is 0 ℃.

Preferably, the fluorescent probe is selected from one or more of a fluorescent protein, a polymer dye or a quantum dot; more preferably, the fluorescent probe is a quantum dot; further preferably, the quantum dots are selected from CdTe, cdSe and CdSe/ZnS core-shell quantum dots, preferably CdSe/ZnS core-shell quantum dots.

Preferably, the quantum dots are selected from one or more of quantum dots with maximum emission wavelength of 525nm-800nm; more preferably, the quantum dots are quantum dots with maximum emission wavelength of 600nm-800nm; most preferably, the quantum dots are quantum dots with a maximum emission wavelength of 655nm.

Preferably, in step 3), the emission wavelength of the image is 525nm to 800nm; preferably, the emission wavelength of the imaging is 600nm to 800nm; most preferably, the emission wavelength of the imaging is 655nm.

Preferably, the excitation wavelength of the imaging is smaller than the emission wavelength of the imaging.

Preferably, a step of centrifuging the streptavidin-containing fluorescent probe is further included before the step 2).

Preferably, the centrifugation is carried out at 3000-7000g for 2-5 minutes; more preferably, the centrifugation is carried out at 5000g for 3-4 minutes.

In another aspect, the invention provides the use of an integrin antibody with a single biotin and a fluorescent probe with streptavidin in the preparation of a kit for detecting epithelial-to-mesenchymal transition of cells.

In yet another aspect, the present invention provides a kit for detecting epithelial-to-mesenchymal transition of a cell, the kit comprising: integrin antibodies with single biotin and fluorescent probes with streptavidin.

Preferably, the integrin antibody with single biotin is selected from one or more of the group consisting of an anti- β 1 integrin antibody with single biotin, an anti- α 5 integrin antibody with single biotin, an anti- β 3 integrin antibody with single biotin, an anti- α v integrin antibody with single biotin; preferably, the integrin antibody with a single biotin is an anti- β 1 integrin antibody or an anti- α 5 integrin antibody with a single biotin; more preferably, the integrin antibody with a single biotin is an anti- β 1 integrin antibody with a single biotin.

Preferably, the fluorescent probe is selected from one or more of a fluorescent protein, a polymer dye or a quantum dot; more preferably, the fluorescent probe is a quantum dot; further preferably, the quantum dots are selected from CdTe, cdSe and CdSe/ZnS core-shell quantum dots, preferably CdSe/ZnS core-shell quantum dots.

Preferably, the quantum dots are selected from one or more of quantum dots with maximum emission wavelength of 525nm-800nm; more preferably, the quantum dots are quantum dots with maximum emission wavelength of 600nm-800nm; most preferably, the quantum dots are quantum dots with a maximum emission wavelength of 655nm. The invention combines integrin kinetics and EMT for the first time and researches, defines the relationship between integrin diffusion movement and cell migration capacity, has important significance for EMT related researches, and can be expanded to other cell research fields, such as researches on the relationship between integrin kinetics and cell morphology or cell adhesion and the like. Compared with the prior art, the invention has at least the following advantages:

1. the method improves the primary antibody and secondary antibody marker integrins in the prior art, adopts quantum dots with stable and durable brightness, and enables a detection system to be more stable, thereby maintaining stable and excellent brightness.

2. Compared with the primary antibody and secondary antibody labeling in the prior art, the method can ensure the relationship between the labeled integrin and 1:1 of the quantum dot, and avoid the condition that one primary antibody is connected with a plurality of secondary antibodies.

3. The method has short period, is faster than the common expression fluorescence mode, and can meet the requirement of rapid determination.

4. The method of the invention is suitable for various cells with different invasion capacities and different mobility capacities.

5. Compared with the traditional mode, the method of the invention does not change the gene in the cell, does not need to kill the cell for fixation, and can still keep the function of the integrin after marking, the marked integrin can participate in the formation of adhesion class, the influence on the cell is small, and the result is more accurate.

6. Compared with the method for measuring the cell morphological change, the method has higher sensitivity and accuracy, and can effectively reduce the false negative rate in the measurement.

7. Compared with the method for measuring cadherin, the method provided by the invention is less in time consumption and simple in experimental steps.

8. The method of the present invention may be applied in labeling various kinds of integrin, and has guiding significance in the research of the diffusion motion of integrin in various kinds of cell.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1A shows that MCF10A cells undergo a gradual change in cell morphology following EMT.

Figure 1B shows the statistical change in cell roundness and area before and after EMT.

FIG. 2A shows a fluorescence image of integrin on the cell surface after labeling taken by a fluorescence microscope.

Fig. 2B shows a diffusion trace plot of a single integrin obtained after continuous shooting for 2000 frames (interval 0.1 s).

FIG. 3A shows the mean MSD diffusion curves for integrin on type E and type M cells and for integrin on MCF7 and MDA-MB-231 cells.

FIG. 3B shows the rate of integrin diffusion on MCF10A, MCF7 and MDA-MB-231 cells before and after EMT induction from the MSD diffusion curve.

FIG. 4 shows a graph of the integrin diffusion rate on cells versus cell roundness and the distribution of the two profiles.

Fig. 5 shows an imaging plot of the α 5 integrin of the quantum dot mark.

FIG. 6A shows fluorescence imaging of quantum dot-labeled integrin and talin protein within the focal adhesion spot by a two-channel imaging system, resulting in a fluorescence-combined image of the two.

Fig. 6B shows the relative light intensity of nascent talin protein at the edge to the quantum dots.

Fig. 7 shows the proportion of the cell fixation trajectories of integrins after Mn-activated quantum dot labeling.

FIG. 8 shows a comparison of images of the primary antibody and secondary antibody labeled with a fluorescent protein of integrin labeled by the method of the present invention.

Figure 9 shows an imaging of cadherin by studying the epithelial-to-mesenchymal transition of cells by a cadherin experiment.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, and the examples are given only for illustrating the present invention and not for limiting the scope of the present invention.

Example 1EMT induction of cells

EMT induction of non-carcinogenic MCF10A cells by addition of TGF-. Beta.1 in the usual manner at 37 ℃ 5% ₂ The cell culture chamber of (2) was incubated for 72 hours or more. As can be seen in fig. 1, the morphology of the cells after EMT induction changed. The cells (cell number) are counted in FIG. 1B>30 Area and roundness), it was found that the average spread area increased and the average roundness decreased. However, the ranges of circularity and spreading area overlap to different degrees, and it is not possible to directly distinguish whether or not the cells have developed EMT.

Wherein the circularity is calculated by the following formula, and as the value approaches 1, it indicates that the cell approaches a circular shape as more:

circularity =4 π area/perimeter ² 。

Example 2Kinetic study of Quantum dot-labeled beta 1 integrin

We used quantum dots to label beta 1-integrin. Labeling experiments were performed on ice to halt the physiological activity of the cells. Cells were first incubated with Anti-ITGB1 (MEM-101), a monoclonal antibody with a single biotin, on ice (about 0 ℃) for 15min to bind specifically to the membrane surface β 1 integrin, and washed with PBS (three times) for use. streptavidin-QD (655nm, invitrogen) was centrifuged at 50rpm for 3 minutes, the supernatant was aspirated, and the above-described conjugate was addedIn the cells after the primary antibody, the cells were incubated on ice (about 0 ℃) for 5 minutes to allow binding. After washing off excess QD (three washes with PBS), the cells were incubated (37 ℃ C., 5% CO) ₂ ) Fluorescence imaging was performed after 15min recovery. The glass culture dish is placed at a 60-fold oil lens, is shot by an EMCCD camera, is excited by 561-nm laser, and obtains a plurality of motion videos of QD-labeled beta 1 integrin by capturing 655nm emitted light signals (the shooting time interval is 10HZ, 2000 frames are shot in a single time, and the shooting time is 30-50 minutes). The movement of β 1 integrin was followed by a Particle tracking plug-in (Particle Tracker) in ImageJ software and diffusion trajectories of greater than 30 frames (3 s) were selected for analysis.

The results are shown in FIGS. 2 and 3. Fig. 2A shows the fluorescence labeling map of the quantum dots we obtained on the surface of a single cell, and fig. 2B shows the diffusion traces of the quantum dot- β 1 integrin 1 obtained after continuous shooting, and the motion information of these traces over time is extracted and analyzed. From the Mean Square Displacement (MSD) curve of the diffusion trace of integrin and the diffusion rate, the diffusion rate of integrin on M-type cells was significantly faster than that of E-type cells. FIG. 3A shows that integrin spreading on M-type cells is higher in MSD curve than E-type cells, and the difference between the two is consistent with the difference between MCF7, a weakly metastatic cancer cell, and MDA-MB-231, a strongly metastatic cancer cell that we used to validate. Fig. 3B shows the diffusion movement velocity calculated from the MSD curve. The movement difference of integrin on E type (epithelial) cells and M type (mesenchymal) cells can be seen more visually. Average diffusion velocity from 0.037 μm ² The/s rises to 0.067 mu m ² A rise of about 80% in the ratio of s, with a median of from 0.03 μm ² The/s rises to 0.06 mu m ² And/s, increased by 100%. Wherein the average maximum diffusion rate of M-type cells is increased by 172% compared to the average minimum diffusion rate of E-type cells. This indicates a significant increase in integrin spreading movements on the cells following EMT induction. Integrin kinetics are distinct on type E and M cells. From the distribution of cell roundness and integrin diffusion rate shown in FIG. 4, the increase in integrin diffusion rate due to EMT induction was independent of cell morphology. Whether in cells with obvious morphological polarity and low roundness or in cells with enclosed morphological polarityObviously, the average diffusion speed of the cells with high roundness is higher than that of the cells with E type. Therefore, the method can be used as a new and more reliable distinguishing mode before and after the EMT induction of the cells.

Example 3Kinetic study of Quantum dot labeled alpha 5 integrin

The experimental conditions were the same as in example 2, except that the monoclonal antibody carrying a single biotin was replaced with an anti- α 5 integrin antibody. The results are shown in FIG. 5. It can be seen that the α 5 integrin labeled with quantum dots is not labeled with β 1 labeled with quantum dots much from the labeling effect, because α 5 can only have one integrin α 5 β 1, but the diffusion movement locus of the integrin can still be well detected.

Example 4Dual channel imaging of integrin and talin proteins

Fluorescence imaging of β 1 integrin and talin protein within the focal adhesion was performed simultaneously. First, GFP-Talin was added to the cell culture solution in the dish and the dish was incubated overnight (12-18 h), and then the integrin was labeled with quantum dots in the manner described in example 2, both were imaged simultaneously, and fluorescence images of both channels were obtained and combined by exciting GFP-Talin with 488nm laser and quantum dots with 561nm laser (see FIG. 6). The movement of the cells was observed by the video of the fluorescence imaging for a long time (2 h), and it could be seen that the labeled integrin reached the most anterior border of the cells and appeared together with the most anterior nascent Talin protein, which indicates that it could participate in the formation of the adhesion spots at the crawling front end of the cells, which indicates that the labeled integrin could still perform the function of integrin, i.e., co-localization with Talin protein, participating in the adhesion spots.

Example 5 Verification experiment 1

The low metastatic cancer cell MCF7 and the high metastatic cancer cell MDA-MB-231 are used for verification. First, as described in example 2, the integrin on two cells was labeled with quantum dots, and fluorescence imaging and analysis were performed to extract the integrin diffusion movement trace and calculate the MSD value. As shown in FIGS. 3A and B, it can be seen that the MSD curve for integrin diffusion on MCF7 cells is lower than that of MDA-MB-231 cells, and thus the diffusion rate is also lower than that of MDA-MB-231 cells. This indicates that the mobility of integrin on MCF7 is weaker than MDA-MB-231. This experiment verifies that the locomotor activity changes of integrins on EMT are true, which indicates that the diffusional motility of integrins on cell membranes is also enhanced when cells acquire high invasiveness or strong locomotor ability. Therefore, there is a positive correlation between the diffusive movement of integrins and the movement of cells.

Example 6 Verification experiment 2

It is known that Mn ions activate integrins to place the integrin in an active conformation, at which time the integrin is immobilized. Mn can therefore be used to verify the function of the labelled integrin. During the imaging of the fluorescence microscope, MCF10A cells are normally photographed and then 0.1mM MnCl is added ₂ And after 10min, shooting is carried out, and counting the proportion of the fixed trace of the integrin on the cells can be seen to be obviously improved (as shown in figure 7), which indicates that the QD-labeled integrin still has an activating function and the labeled integrin can participate in the formation of an agglutinate.

Comparative example 1Labeling integrin by primary antibody and secondary antibody with fluorescent protein

The experimental conditions were the same as those of example 2, except that the primary antibody was used to wash off excess antibody, which was then linked to a secondary antibody with green fluorescent protein Alex488 (GFP), and the secondary antibody was imaged with 488nm excitation light and analyzed. The results are shown in fig. 8, the left image is the imaging result of the comparative example, and the right image is the imaging result of the quantum dot labeled integrin of the present invention. It can be seen that the imaging graph (left graph) of the fluorescent protein labeled integrin is unclear and cannot distinguish single integrin, while the imaging graph (right graph) of the quantum dot labeled integrin of the invention is very clear and can clearly distinguish single integrin, thereby realizing the analysis of the diffusion trace of the single integrin.

Comparative example 2Study of epithelial-mesenchymal transition of cells by cadherin assay (immunostaining)

Cells were fixed at room temperature for 1h (4% formaldehyde solution), washed with PBS and then punched for 10min (0.2% Triton X-100). After washing with PBS, 1% BSA was added and incubated for 1h, and then anti-E-cadherin monoclonal antibody was added and incubated overnight (12h, 4 ℃). The following day, washing with PBS solution, alex 488-anti mouse IgG (1 h, room temperature) was added, phalloidin-594 was added to stain actin, washing with PBS was performed, and imaging was performed, and the results are shown in FIG. 9. The results show a decrease in the adhesion protein cadherin at the cell-to-cell boundary after EMT (right panel). Although it is possible to separate E-type cells and M-type cells, this method requires fixed cells, cannot perform a live cell experiment, takes a long time, requires overnight (more than 12 hours), is complicated in procedure, and is not suitable for rapid analysis of the state of cells. The method of the invention is carried out in living cells, and the time consumption is usually less than 1 hour, thereby greatly improving the detection efficiency.

Claims (10)

1. A method of detecting epithelial-to-mesenchymal transition of a cell, the method comprising the steps of:

1) Incubating the integrin antibody with single biotin with the cells to be detected to specifically recognize and bind integrin on the cell membrane surface of the cells, and removing unbound integrin antibody with single biotin after the incubation is completed;

2) Adding a fluorescent probe with streptavidin into the obtained substance in the step 1), incubating, and removing the unreacted redundant fluorescent probe with streptavidin after reaction to obtain the integrin marked by the fluorescent probe;

3) Detecting a change in the average diffusion rate of the fluorescent probe-labeled integrin relative to control cells by imaging the fluorescent probe for the fluorescent probe-labeled integrin obtained in step 2), wherein an increase in the average diffusion rate of the fluorescent probe-labeled integrin relative to control cells indicates epithelial-to-mesenchymal transition of the cells.

2. The method of claim 1, wherein the mean diffusion rate of the fluorescent probe-labeled integrin is increased by 40% -172% relative to control cells; preferably, the average diffusion rate of the fluorescent probe-labeled integrin is increased by 80% to 172% relative to control cells.

3. The method according to claim 1 or 2, wherein step 1) is preceded by a step of pre-treating the cells;

preferably, the cells are selected from one or more of epithelial cells, stromal cells, tumor cells, peripheral blood mononuclear cells or lymphocytes; more preferably, the cells are epithelial cells or mesenchymal cells; most preferably, the cell is a normal breast cell MCF10A or a breast cell MCF10A after epithelial-mesenchymal transition;

preferably, the control cells are selected from the group consisting of corresponding cells that have not undergone epithelial-to-mesenchymal transition; more preferably, the control cell is an epithelial cell; most preferably, the control cell is a normal breast cell MCF10A.

4. The method according to any one of claims 1-3, wherein in step 1), the incubation time is 10 to 18 minutes, the incubation temperature is 0 to 4 ℃; more preferably, the incubation time is 15 minutes and the incubation temperature is 0 ℃;

preferably, the integrin antibody with single biotin is selected from one or more of an anti- β 1 integrin antibody with single biotin, an anti- α 5 integrin antibody with single biotin, an anti- β 3 integrin antibody with single biotin, an anti- α v integrin antibody with single biotin; preferably, the integrin antibody with a single biotin is an anti- β 1 integrin antibody or an anti- α 5 integrin antibody with a single biotin; more preferably, the integrin antibody with a single biotin is an anti- β 1 integrin antibody with a single biotin.

5. The method according to any one of claims 1-4, wherein in step 2), the incubation time is 3 to 8 minutes and the incubation temperature is 0 to 4 ℃; more preferably, the incubation time is 5 minutes and the incubation temperature is 0 ℃;

preferably, the fluorescent probe is selected from one or more of a fluorescent protein, a polymer dye or a quantum dot; more preferably, the fluorescent probe is a quantum dot; further preferably, the quantum dots are selected from CdTe, cdSe and CdSe/ZnS core-shell quantum dots, preferably CdSe/ZnS core-shell quantum dots;

preferably, the quantum dots are selected from one or more of quantum dots with maximum emission wavelength of 525nm-800nm; more preferably, the quantum dots are quantum dots with maximum emission wavelength of 600nm-800nm; most preferably, the quantum dots are quantum dots with a maximum emission wavelength of 655nm.

6. The method of any one of claims 1-5, wherein in step 3) the imaged emission wavelength is from 525nm to 800nm; preferably, the emission wavelength of the imaging is 600nm to 800nm; most preferably, the emission wavelength of the imaging is 655nm.

7. The method of any one of claims 1-6, further comprising the step of centrifuging the streptavidin-bearing fluorescent probe prior to step 2);

preferably, the centrifugation is carried out at 3000-7000g for 2-5 minutes; more preferably, the centrifugation is carried out at 5000g for 3-4 minutes.

8. Use of an integrin antibody with single biotin and a fluorescent probe with streptavidin for the preparation of a kit for detecting epithelial-to-mesenchymal transition of cells.

9. A kit for detecting epithelial-to-mesenchymal transition of a cell, the kit comprising: integrin antibodies with single biotin and fluorescent probes with streptavidin.

10. The kit of claim 9, wherein the integrin antibody with single biotin is selected from one or more of an anti- β 1 integrin antibody with single biotin, an anti- α 5 integrin antibody with single biotin, an anti- β 3 integrin antibody with single biotin, an anti- α v integrin antibody with single biotin; preferably, the integrin antibody with a single biotin is an anti- β 1 integrin antibody or an anti- α 5 integrin antibody with a single biotin; more preferably, the integrin antibody with a single biotin is an anti- β 1 integrin antibody with a single biotin;

preferably, the fluorescent probe is selected from one or more of a fluorescent protein, a polymer dye or a quantum dot; more preferably, the fluorescent probe is a quantum dot; further preferably, the quantum dots are selected from CdTe, cdSe and CdSe/ZnS core-shell quantum dots, preferably CdSe/ZnS core-shell quantum dots;

preferably, the quantum dots are selected from one or more of quantum dots with maximum emission wavelength of 525nm-800nm; more preferably, the quantum dots are quantum dots with maximum emission wavelength of 600nm-800nm; most preferably, the quantum dots are quantum dots with a maximum emission wavelength of 655nm.

Images