CN108753573A - The method for being captured in micro-fluidic chip and identifying fetal nucleated red blood - Google Patents

Abstract

The present invention provides a method for capturing and identifying fetal nucleated erythrocytes in a microfluidic chip, comprising: Step 1. Synthesizing gelatin nanoparticles by a two-step desolventization method and preparing a precursor solution, and then passing it into a microfluidic chip, Its inner wall forms a nano-coating, in which the microfluidic chip contains multiple serpentine main channels and multiple fishbone units; step 2. Use chemical modification methods to modify streptavidin on the nano-coating, and then graft biological The antibody modified with protein; Step 3. Use the density gradient centrifugation method to separate the mononuclear cell layer containing fetal nucleated red blood cells from the peripheral blood; Step 4. Pass the mononuclear cell suspension into the microfluidic chip at a certain flow rate for capture Step 5. Pass a certain concentration of gelatinase into the microfluidic chip that captures fetal nucleated red blood cells, and release fetal nucleated red blood cells by degrading the nano-coating; Step 6. Use specific antibodies to capture fetal nucleated red blood cells; Nuclear red blood cells were identified.

Description

Method for capturing and identifying fetal nucleated red blood cells in a microfluidic chip

technical field

The invention belongs to the field of fetal cell sorting and identification methods, and in particular relates to a method for capturing and identifying fetal nucleated red blood cells in a microfluidic chip.

Background technique

Fetal cells can enter the maternal peripheral blood through the placental barrier during pregnancy. Fetal cells have a variety of characteristics: ① belong to monocytes and contain all the genetic information of the fetus; ② have specific proteins on the surface that are different from normal blood cells; ③ have a short cycle in the peripheral blood of pregnant women, and are not affected by previous diagnosis when used for diagnosis. effects of pregnancy. The special properties of fetal cells provide new ideas for non-invasive prenatal diagnosis, and many studies are devoted to the enrichment of fetal cells. Due to the extremely small number of fetal cells in the peripheral blood of pregnant women, it has always been a technical problem to efficiently sort them.

Traditional cell sorting techniques include magnetic sorting and flow cytometry. Magnetic separation technology is based on the effect of an external magnetic field, using specific antibody-labeled magnetic particles to separate target cells. This method is simple to operate and takes less time, but has the problem of low separation purity. The equipment used in flow cytometry is expensive, and requires professionals to operate. At the same time, it requires a certain amount of cells, which limits its popularization in the laboratory. In recent years, the use of microfluidic chips combined with three-dimensional nanomaterials to capture rare cells has become more and more widely used. Microfluidic technology mainly integrates the functions of sample pretreatment, sample manipulation, sample reaction and signal detection on a microchip to achieve portable, fast, real-time and efficient detection and analysis. Due to its special size and structure, three-dimensional nanomaterials exhibit characteristics such as small size effect and high specific surface area, which can increase the contact probability between the cells to be tested and the target. At the same time, the rough surface of nanomaterials can reduce electrostatic force and thus reduce repulsion It can increase the adhesion of target cells and greatly improve the enrichment efficiency of cells to be tested. The modification of nanomaterials to the inner wall of the channel of the microfluidic chip greatly enhances the adsorption ability of the chip to the target cells, showing an efficient capture ability.

In addition, since pregnant women's own nucleated red blood cells will express γ-globin, the traditional use of γ-globin antibodies with fluorescent groups cannot distinguish the fetal origin of nucleated red blood cells.

Contents of the invention

The present invention is made to solve the above problems, and aims to provide a method for capturing and identifying fetal nucleated red blood cells in the peripheral blood of pregnant women in a microfluidic chip with high efficiency and high specificity.

In order to achieve the above object, the present invention adopts the following means.

The invention provides a method for capturing and identifying fetal nucleated red blood cells in a microfluidic chip, which is characterized in that it comprises the following steps: Step 1. Synthesize gelatin nanoparticles by a two-step desolventization method and prepare them into a precursor solution. The liquid is passed into the microfluidic chip, and a nano-coating is formed on the inner wall of the channel of the microfluidic chip. The microfluidic chip includes: a plurality of serpentine main channels (serpentine channels), and a A plurality of fishbone units arranged along the flow direction above the channel, each fishbone unit extends along the width direction of the serpentine main channel and is L-shaped; Step 2. Use chemical modification methods to modify the nano-coating Add strontavidin, and then graft biotin-modified antibody; Step 3. Use density gradient centrifugation to separate the mononuclear cell layer containing fetal nucleated erythrocytes from peripheral blood; Step 4. Mononuclear cell suspension Enter the microfluidic chip at a certain flow rate of 0.4-1.6mL/h for capture; Step 5. Pass a certain concentration of gelatinase into the microfluidic chip that captures fetal nucleated red blood cells, and release fetal erythrocytes by degrading the nano-coating. nucleated erythrocytes; and step 6. identifying the captured fetal nucleated erythrocytes using specific antibodies.

Preferably, in the method for capturing and identifying fetal nucleated red blood cells in the microfluidic chip involved in the present invention, it may also have such a feature: the length from the inlet end to the outlet end of the microfluidic chip is 35 mm, and each snake The distance between the inflow end and the outflow end of the shaped main channel is 22mm, so that the setting effect is better.

Preferably, in the method for capturing and identifying fetal nucleated red blood cells in the microfluidic chip involved in the present invention, it may also have such a feature: the microfluidic chip contains six serpentine main channels arranged in parallel, each The main part of the serpentine main channel is composed of semicircular rings that protrude upwards and downwards alternately and extend continuously. The inner diameter of each semicircular ring is 200 μm and the outer diameter is 500 μm. The width of the serpentine main channel is 300μm, the depth is 50μm, so the setting effect is better.

Preferably, in the method for capturing and identifying fetal nucleated red blood cells in the microfluidic chip involved in the present invention, it also has such a feature: all fishbone units are arranged at intervals of 45°, and each fishbone unit contains two The two sides are arranged at an angle of 90°, the longitudinal height of the fishbone unit is 35 μm, and the width is 50 μm, so that the setting effect is better.

Preferably, in the method for capturing and identifying fetal nucleated erythrocytes in the microfluidic chip involved in the present invention, it may also have such a feature: in step 1, the particle diameter of the gelatin nanoparticles is 200nm, and the The concentration is 2.0mg/mL. In step 1, the method of obtaining the nano-coating is as follows: gelatin nanoparticles are purified and then freeze-dried, washed with morpholineethanesulfonic acid (MES) solution, and reacted in the EDC/NHS system to a certain extent. time to obtain NHS-modified nanoparticles; then disperse the NHS-modified nanoparticles in deionized water to prepare a functionalized precursor; pass 3-aminopropyltriethoxysilane into the microfluidic chip, and the The inner wall forms an aminated surface; finally, the precursor solution is passed into the aminated microfluidic chip, and incubated for a certain period of time to form a nano-coating.

Preferably, in the method for capturing and identifying fetal nucleated erythrocytes in the microfluidic chip involved in the present invention, it may also have such a feature: Step 2 is specifically: first rinse the microfluidic chip with phosphate buffer solution completely Afterwards, 20-100 μg/mL streptavidin was injected, overnight at 4°C, after washing, 10-50 μg/mL anti-CD147 antibody was injected and left at room temperature for 1-3 hours. The optimal concentration of streptavidin is 50 μg/mL; the optimal concentration of anti-CD147 antibody is 20 μg/mL, and the storage time is 2 hours.

Preferably, in the method for capturing and identifying fetal nucleated erythrocytes in the microfluidic chip involved in the present invention, it may also have such a feature: step 3 is specifically: first slowly add the blood sample to the lymphocyte separation solution Then centrifuge at 400g for 30 minutes, and take out the mononuclear cell layer after the blood is layered.

Preferably, in the method for capturing and identifying fetal nucleated red blood cells in the microfluidic chip of the present invention, it may also have such a feature: in step 4, the flow rate is 0.4 mL/h.

Preferably, in the method for capturing and identifying fetal nucleated red blood cells in the microfluidic chip involved in the present invention, it may also have such a feature: in step 5, the gelatinase used is matrix metalloproteinase MMP-9, And the concentration is 0.01-0.1 mg/mL, preferably 0.1 mg/mL.

Preferably, in the method for capturing and identifying fetal nucleated red blood cells in the microfluidic chip involved in the present invention, it may also have such a feature: Step 6 is specifically: for the captured fetal nucleated red blood cells, sequentially use 4 wt .% paraformaldehyde solution for 10min, 0.1wt.% polyethylene glycol octyl phenyl ether (triton X-100) solution for 10min perforation, 3wt.% bovine serum albumin (BSA) blocking solution for 30min ; Then add specific immunofluorescent protein dyes anti-ε-globin-PE and anti-CD45-FITC, and store overnight at 4°C in the dark; finally use 4',6-diamidino-2-phenylindole (DAPI) to counterstain the nuclei and observe the results under a fluorescent microscope.

Function and Effect of Invention

(1) The microfluidic chip adopts a serpentine main channel combined with a fishbone unit, which increases the collision probability between the cells in the fluid and the inner wall of the channel, thereby enhancing the capture effect.

(2) The method of forming a nano-coating on the inner wall of the channel through electrostatic adsorption is simple to operate and low in price, and the synthetic particle size can be adjusted to achieve different roughness;

(3) The rough surface of the gelatin nanocoating not only increases the grafting rate of the antibody but also enhances the cell attachment behavior, and the biodegradable property of the gelatin is conducive to the non-destructive release of the target cells;

(4) Using the protein ε-globulin unique to fetal nucleated red blood cells, the fetal origin of the cells can be accurately judged;

(5) The structure of the microfluidic chip used in this scheme is simple and convenient, and the whole scheme has the advantages of easy operation, strong specificity, and low cost, so it has a good application prospect.

Description of drawings

1 is a schematic diagram of the process of capturing fetal nucleated red blood cells in a microfluidic chip according to an embodiment of the present invention;

Fig. 2 is the TEM characterization diagram of the gelatin nanoparticles involved in the embodiment of the present invention;

Fig. 3 is an AFM characterization diagram of the nano-coating formed on the inner wall of the channel of the microfluidic chip according to the embodiment of the present invention;

Fig. 4 is a physical structure diagram of the microfluidic chip involved in the embodiment of the present invention;

Fig. 5 is an enlarged view of a part of the serpentine main channel and the fishbone unit in the microfluidic chip according to the embodiment of the present invention taken under an optical microscope;

Fig. 6 is a cross-sectional view of the microfluidic chip involved in the embodiment of the present invention;

Fig. 7 is a bright-field effect diagram of the microfluidic chip according to the embodiment of the present invention capturing fetal nucleated red blood cells in the peripheral blood of pregnant women;

Fig. 8 is a SEM characterization diagram of fetal nucleated red blood cells captured by the microfluidic chip involved in the embodiment of the present invention in the peripheral blood of pregnant women;

Fig. 9 is a graph showing the concentration of gelatin nanoparticles in the microfluidic chip involved in the embodiment of the present invention on the capture efficiency of nucleated erythrocytes;

Fig. 10 is a bright field effect diagram of the matrix metalloproteinase MMP-9 involved in the embodiment of the present invention after releasing the cells captured on the microfluidic chip;

Figure 11 is a diagram of the activity test results of the matrix metalloproteinase MMP-9 involved in the embodiment of the present invention after releasing the cells captured on the microfluidic chip;

Fig. 12 is a result diagram of identifying fetal nucleated erythrocytes using specific fluorescent proteins in the embodiment of the present invention;

Fig. 13 is a schematic diagram of the statistical results of fetal nucleated red blood cells captured and identified from the peripheral blood of pregnant women in the embodiment of the present invention.

Detailed ways

The method for capturing and identifying fetal nucleated red blood cells in the microfluidic chip involved in the present invention will be described in detail below with reference to the accompanying drawings.

<Example 1>

As shown in Figure 1, the specific steps of the method for capturing and identifying fetal nucleated red blood cells in the microfluidic chip provided in this embodiment are:

(1) Preparation of gelatin nanoparticles:

Weigh 0.625g B-type gelatin and dissolve it in 12.5mL deionized water, heat to dissolve at 50°C. Then 12.5 mL of acetone was added at a rate of 6 mL/h at 300 rpm, and after standing for 1 min, the supernatant was discarded. Add 12.5 mL of deionized water to the precipitate, and heat again at 50°C to dissolve. Use 2mol/L NaOH solution to adjust the pH of the reactant to 10, then slowly add about 45mL of acetone to the gelatin solution, and finally add glutaraldehyde (1.5%) solution, in situ cross-linking for 2 hours, and stop cross-linking by excess glycine couplet. The gelatin nanoparticles were recovered by centrifuging the above reaction solution at 10000 rpm for 10 min. Freeze-drying is carried out by a freeze dryer to remove residual organic matter in the sample. As shown in Figure 2, the morphology of nanoparticles can be clearly seen, with a size of about 200nm.

(2) Preparation of nano-coating inside the microfluidic chip and antibody modification

Inject an absolute ethanol solution containing 5 wt.% silane (APTES) into the microfluidic chip, incubate at room temperature for 1 hour, and introduce amino-NH ₂ into the inner wall of the microfluidic chip. After the gelatin nanoparticles were washed with morpholineethanesulfonic acid (MES) solution, they were reacted in 200 μL of a mixture of 4 mg/mL EDC and 6 mg/mL NHS for 30 min, and the -NHS group was introduced on the surface of the gelatin particles using the EDC-NHS reaction system , and the -NHS-modified gelatin nanoparticle solution (2 mg/mL) was passed into the aminated microfluidic chip, incubated for 30 min, and a layer of nano-coating was formed on the inner wall of the channel by electrostatic adsorption. As shown in Figure 3, it can be seen that the nano-coating forms a roughness of about 120nm.

Then, 50 μg/mL streptavidin was introduced into the microfluidic chip with gelatin nano-coating to react with the remaining -NHS groups on the surface of the gelatin nanoparticles, at 4°C overnight. Then a biotinylated 20 μg/mL CD147 antibody was passed through, and the antibody was modified into the microfluidic chip by using the force between streptavidin and biotin.

In this embodiment, the microfluidic chip used is manufactured according to traditional photolithography technology, and its structure is shown in Figures 4 to 6. The microfluidic chip 10 includes an inlet port 11, an outlet port 12, and six serpentine main grooves. Road 13, a plurality of herringbone units 14, and three rows of drainage channels 15.

The six serpentine main channels 13 are arranged parallel to each other, and are arranged in groups of two by two, and the two ends (inflow end and outflow end) of each group of serpentine main channels 13 are connected correspondingly, and the three groups are connected in parallel with each other, and pass through the intersecting channels respectively. The three rows of drains 15 communicate with the inlet port 11 and the outlet port 12 .

As shown in FIGS. 3 and 4 , the main body of each serpentine main channel 13 is composed of semicircular rings that protrude upwards and downwards alternately and extend continuously. In this embodiment, the inner diameter of each semi-circular ring is 200 μm, the outer diameter is 500 μm, the width of the serpentine main channel 13 is 300 μm, and the depth H1 is 50 μm; the length from the inlet end to the outlet end of the microfluidic chip 10 is 35 mm , the linear distance between the inflow end and the outflow end of each serpentine main channel 13 is 22 mm.

The herringbone unit 14 is embedded above the serpentine main channel 13 as an eddy current generating functional unit, all herringbone units 14 are arranged along the flow direction, and the adjacent herringbone units 14 are spaced at an angle of 45° (based on the angle of the serpentine main channel 13 The semicircle is the center of the circle). Each fishbone unit 14 extends along the width direction of the serpentine main channel 13, and each fishbone unit 14 includes two side parts, the two side parts are arranged at an angle of 90°, and the whole is L-shaped. In this embodiment, the longitudinal height H2 of the fishbone unit 14 is 35 μm, and the width is 50 μm.

Using the microfluidic chip 10 with the above structure can better ensure the optimal effect of this solution.

<Example 2>

Dilute 2mL of maternal peripheral blood with PBS at a ratio of 1:1, then place it on a 4mL lymphocyte separation medium, centrifuge at 400g for 30min, obtain a mononuclear layer containing fetal nucleated red blood cells, and make 1mL of cell suspension. Then pass through the antibody-modified microfluidic chip 10, under the action of the fluid, the background interfering cells in the suspension are recovered from the outlet port along with the fluid, while the fetal nucleated red blood cells remain in the microfluidic chip through the specific recognition of the antigen antibody 10 internal, to achieve capture. As shown in FIG. 7 , after the capture is completed, the fetal nucleated red blood cells are captured on the inner wall of the microfluidic chip 10 . As shown in Figure 8, the cells spread out on the surface of the nano-coating and are firmly adsorbed on the surface of the nano-coating.

<Example Three>

To explore the effect of the concentration of gelatin nanoparticles on the efficiency of target cell sorting, the specific operation is: use different concentrations (1.0mg/mL, 1.5mg/mL, 2.0mg/mL, 2.5mg/mL) of gelatin nanoparticles in the microfluidic A nano-coating is formed inside the control chip 10 and modified with antibodies. A 1 mL sample of nucleated erythroid TF-1 cells at a concentration of 200/mL was passed into the microfluidic chip 10 to complete capture. Observing and counting under the Olympus IX 81 microscope, the experimental results are shown in Figure 9, when the concentration of gelatin nanoparticles is 2.0mg/mL, the capture efficiency is 81%. <Example 4>

A matrix metalloproteinase (MMP-9) solution is introduced into the microfluidic chip 10 that has completed the capture of fetal nucleated red blood cells to degrade the gelatin nanocoating on the surface of the channel, thereby realizing the release of fetal nucleated red blood cells. Collect target cells at the outlet port. As shown in FIG. 10 , it can be seen that after degrading the gelatin, the cells fall off from the inner wall of the microfluidic chip 10 .

<Embodiment 5>

Viability characterization of recovered fetal nucleated erythrocytes. Collect the cells at the bottom of the test tube by centrifugation, add the active dye FDA/PI mixture, incubate in the dark for 10 minutes, and then wash with PBS for 3 minutes. Cell viability was observed under an Olympus IX 81 microscope. Active cells appear green under the action of FDA, and apoptotic cells appear red under the action of PI. The effect is shown in Figure 11, the cells maintain good activity after enzymatic release.

<Example 6>

Immunofluorescence identification of released and recovered fetal nucleated erythrocytes: first step: fixation. The cells were fixed with 4wt.% paraformaldehyde (PFA) solution to keep the cells in a good shape, reacted for 10 minutes, and washed once with PBS; the second step: perforation. The cells were perforated with 0.1wt.% Triton-X100 solution, so that the dye molecules could enter the cytoplasm, reacted for 10 minutes, and washed once with PBS; the third step: blocking. Block with 10wt.% goat serum for 30 minutes, wash with PBS; step 4: staining. Add 10 microliters each of anti-CD45-FITC and anti-ε-globin-PE fluorescent dyes for immunofluorescent staining of the cells, and react overnight at 4°C, and finally use DAPI dyes for nuclear staining. Observed under Olympus IX 81 microscope, anti-CD45-FITC emits green fluorescence under blue light excitation for identifying white blood cells, anti-ε-globin-PE emits red fluorescence under green light excitation for identifying fetal nucleated red blood cells, and DAPI in Under the excitation of ultraviolet light, it turns blue and is used for cell nucleus staining (Figure 12). Comprehensive results: (1) fetal nucleated red blood cells (ε-globin+/CD45-/DAPI+), (2) white blood cells (ε-globin-/CD45+/DAPI+).

<Embodiment 7>

Detection of fetal nucleated red blood cells in the peripheral blood of pregnant women: 12 cases of peripheral blood of pregnant women at 7 to 13 weeks were collected for the sorting and identification of fetal nucleated red blood cells. The capture of target cells was completed according to Example 2, and the fluorescence identification of target cells was completed according to Example 5, and the number of captured and identified target cells was counted (as shown in FIG. 13 ). The number of fetal nuclear red captured from the peripheral blood of pregnant women in the early pregnancy is 3-24/mL, and fluctuates with the gestational age.

The above is only an illustration of the technical solution of the present invention. The method for capturing and identifying fetal nucleated red blood cells in the microfluidic chip involved in the present invention is not limited to the structure described above, but is subject to the scope defined in the claims. Any modifications, supplements or equivalent replacements made by those skilled in the art of the present invention on this basis are within the scope of protection required by the claims of the present invention.

Claims (10)

1. A method for capturing and identifying fetal nucleated red blood cells in a microfluidic chip, characterized in that, comprising the steps: Step 1. Synthesize gelatin nanoparticles by a two-step desolventization method and prepare a precursor solution, pass the precursor solution into a microfluidic chip, and form a nanocoating on the inner wall of the channel of the microfluidic chip, wherein the microfluidic chip contains : a plurality of serpentine main channels, and a plurality of herringbone units arranged above the serpentine main channel and arranged along the flow direction, each herringbone unit extends along the width direction of the serpentine main channel and is L shape; Step 2. Using a chemical modification method to modify streptavidin on the nano-coating, and then grafting biotin-modified antibodies; Step 3. Using density gradient centrifugation to separate the mononuclear cell layer containing fetal nucleated erythrocytes from peripheral blood; Step 4. Pass the mononuclear cell suspension into the microfluidic chip at a certain flow rate of 0.4-1.6mL/h for capture; Step 5. Passing a certain concentration of gelatinase into the microfluidic chip capturing fetal nucleated red blood cells, releasing the fetal nucleated red blood cells by degrading the nanocoating; and Step 6. Using specific antibodies to identify the captured fetal nucleated erythrocytes. 2. the method for capturing and identifying fetal nucleated red blood cells in a microfluidic chip according to claim 1, characterized in that: Among them, the microfluidic chip contains six serpentine main channels arranged in parallel, The main part of each serpentine main channel is composed of semicircular rings that protrude upwards and downwards alternately and extend continuously. The inner diameter of each semicircular ring is 200 μm and the outer diameter is 500 μm. The width is 300 μm and the depth is 50 μm. 3. the method for capturing and identifying fetal nucleated red blood cells in a microfluidic chip according to claim 1, characterized in that: Wherein, the length from the inlet end to the outlet end of the microfluidic chip is 35 mm, and the distance between the inflow end and the outflow end of each serpentine main channel is 22 mm. 4. the method for capturing and identifying fetal nucleated red blood cells in a microfluidic chip according to claim 1, characterized in that: Among them, all fishbone units are arranged at intervals of 45°, Each fishbone unit contains two sides, and the two sides are set at an angle of 90°. The longitudinal height of the fishbone unit is 35 μm and the width is 50 μm. 5. the method for capturing and identifying fetal nucleated red blood cells in a microfluidic chip according to claim 1, characterized in that: Wherein, in step 1, the particle diameter of gelatin nanoparticles is 200nm, and the concentration of precursor solution is 2.0mg/mL, In step 1, the method for obtaining the nano-coating is: purifying the gelatin nanoparticles, freeze-drying, washing with morpholineethanesulfonic acid solution, and reacting in the EDC/NHS system for a certain period of time to obtain NHS-modified nanoparticles; Then the NHS-modified nanoparticles were dispersed in deionized water to prepare a functionalized precursor solution; 3-aminopropyltriethoxysilane was passed into the microfluidic chip to form an aminated surface on the inner wall of the chip; finally the precursor solution Pass through the aminated microfluidic chip and incubate for a certain period of time to form a nano-coating. 6. the method for capturing and identifying fetal nucleated red blood cells in a microfluidic chip according to claim 1, characterized in that: Among them, step 2 is as follows: first rinse the microfluidic chip with phosphate buffer solution completely, then inject 20-100 μg/mL streptavidin, overnight at 4°C, and inject 10-50 μg/mL after washing anti-CD147 antibody and placed at room temperature for 1 to 3 hours. 7. the method for capturing and identifying fetal nucleated red blood cells in a microfluidic chip according to claim 1, characterized in that: Wherein, step 3 specifically includes: first slowly drop the blood sample onto the liquid surface of the lymphocyte separation solution, then centrifuge at 400 g for 30 minutes, and take out the mononuclear cell layer after the blood is separated. 8. the method for capturing and identifying fetal nucleated red blood cells in a microfluidic chip according to claim 1, characterized in that: Wherein, in step 4, the flow rate is 0.4mL/h. 9. the method for capturing and identifying fetal nucleated red blood cells in a microfluidic chip according to claim 1, characterized in that: Wherein, in step 5, the gelatinase used is matrix metalloproteinase 9, and the concentration is 0.01-0.1 mg/mL. 10. the method for capturing and identifying fetal nucleated red blood cells in a microfluidic chip according to claim 1, characterized in that: Among them, step 6 is specifically: for the captured fetal nucleated red blood cells, use 4wt.% paraformaldehyde solution to fix for 10 minutes, 0.1wt.% polyethylene glycol octylphenyl ether solution to perforate for 10 minutes, 3wt.% Blocked with bovine serum albumin blocking solution for 30 minutes; then added specific immunofluorescent protein dyes anti-ε-globin-PE and anti-CD45-FITC, and stored overnight at 4°C in the dark; finally used 4',6- The nuclei were counterstained with diamidino-2-phenylindole, and the results were observed under a fluorescent microscope.