CN114774467B

CN114774467B - Preparation method of multiple fluorescent marked living cell sample

Info

Publication number: CN114774467B
Application number: CN202210369640.4A
Authority: CN
Inventors: 付玲; 黄歆媛; 高秀娟; 陈忠云
Original assignee: Hubei Optics Valley Laboratory; Huazhong University of Science and Technology
Current assignee: Hubei Optics Valley Laboratory; Huazhong University of Science and Technology
Priority date: 2022-04-08
Filing date: 2022-04-08
Publication date: 2024-03-29
Anticipated expiration: 2042-04-08
Also published as: CN114774467A

Abstract

The invention relates to a preparation method of a multiple fluorescent labeling living cell sample, based on the method provided by the invention, nine fluorophores which can be used simultaneously and can be used for realizing resolution are screened, a subcellular structure labeling strategy is designed and optimized, a specific two-photon selective excitation fluorescent microscopic imaging system is combined, a direct fluorescent labeling and fluorescent protein co-localization technology is combined, nine fluorescent labeling can be realized in the living cell imaging sample at the same time, nine target structures are observed simultaneously in one-time imaging, and at most nine-color living cell fluorescent microscopic imaging is realized.

Description

Preparation method of multiple fluorescent marked living cell sample

Technical Field

The invention belongs to the technical field of biological markers, and particularly relates to a preparation method of a multiple fluorescent marked living cell sample.

Background

With the development of fluorescent probes and biomarker techniques, researchers can use fluorescent microscopy imaging techniques to monitor information about molecules in living cells in real time. The application of multiple fluorescent markers combined with fluorescent multicolor microscopic imaging to cytology can reveal the interaction among organelles, monitor the number, volume and spatial distribution of specific organelles in living cells in real time, and monitor the change of molecular concentration participating in biochemical reaction qualitatively or quantitatively.

The fluorescent molecular probes currently applied to living cells and living body detection mainly comprise: gene-encoded probes (e.g., fluorescent proteins and luciferases), small organic molecule fluorescent dye sets, and fluorescent nanoparticles. Common fluorescent labeling techniques include direct fluorescent labeling, immunofluorescent labeling, and fluorescent protein co-localization techniques. The direct fluorescent labeling refers to covalent bonding of fluorescent dye molecules and a labeled substance, and the method has simple experimental operation, but is difficult to realize specific labeling; the immunofluorescence labeling is to realize the specificity labeling of the specificity antibody coupled with the fluorescent dye in situ of the tissue cells through antigen-antibody reaction, so that the experimental operation is more complicated and the experimental cost is higher; the fluorescent protein co-localization technology is to design a proper plasmid to realize the fusion of fluorescent protein and the target protein to be marked, and then introduce DNA into cells by means of transfection technology to realize fluorescent expression, thereby realizing specific marking. In order to explore life process at cell level and explore interactions among different substances and organelles in cells, multiple specific fluorescent markers are needed to be realized in a living cell state, the types of fluorophores capable of realizing specific markers simultaneously in living cell samples reported at home and abroad are not more than 6, and the quantity of fluorescent marker substances and organelles is limited.

Disclosure of Invention

The technical problems solved by the invention are as follows: the preparation method of the multiple fluorescent labeling living cell sample is provided, so that the number of target types which can be observed in the living cell sample at the same time is increased to nine, and nine-color fluorescent microscopic imaging of the living cells is realized.

The specific solution provided by the invention is as follows:

the invention provides a preparation method of a multiple fluorescent marked living cell sample, which comprises the following steps:

s1, gene fusion: respectively connecting a dark blue fluorescent protein EBFP2.0 gene with a fibrin gene to form a recombinant EBFP2.0 gene, connecting a red fluorescent protein mKate gene with a pyruvate dehydrogenase (pyruvate dehydrogenase gene) to form a recombinant mKate gene, connecting a blue fluorescent protein TagBFP gene with a histone gene to form a recombinant TagBFP gene, connecting a green fluorescent protein mAmetrine gene with a reticulin gene to form a recombinant mAmetrine gene, connecting a cyan fluorescent protein mCerulean gene with a beta-actin gene to form a recombinant mCerulean gene, and connecting a green fluorescent protein mClover3 gene with a sialyltransferase (SiT-15) gene to form a recombinant mClover3 gene;

s2, constructing plasmids: inserting the recombinant mCerulean gene and the recombinant mKate gene into a eukaryotic expression vector to construct a first plasmid for coexpression of cyan fluorescent protein mCerulean and red fluorescent protein mKate; inserting the recombinant tagBFP gene and the recombinant mAmetrine gene into a eukaryotic expression vector to construct a second plasmid for co-expressing the blue fluorescent protein tagBFP and the green fluorescent protein mAmetrine; inserting the LSSmCherry gene and the recombinant mClover3 gene into a eukaryotic expression vector to construct a third plasmid for coexpression of the red fluorescent protein LSSmCherry and the green fluorescent protein mClover 3; the EBFP2.0 gene was inserted into a eukaryotic expression vector to construct a fourth plasmid for expressing the dark blue fluorescent protein EBFP 2.0.

S3, cell transfection: co-transfecting a first target cell with the first plasmid and the second plasmid by adopting a liposome transfection reagent to obtain a first cell strain capable of expressing fluorescent protein mCerulean, mKate, tagBFP and mAmetrine, and co-transfecting a second target cell with the third plasmid and the fourth plasmid to obtain a second cell strain capable of expressing fluorescent protein LSSmCherry, mClover and EBFP 2.0;

s4, cell CFSE staining: performing direct fluorescent labeling on the third target cells by using a first fluorescent dye CFSE to obtain a third cell strain;

s5, mixed inoculation: respectively subjecting the first cell strain, the second cell strain and the third cell strain to trypsin digestion, mixing after digestion, and then culturing for 5-12 hours to obtain a fourth cell strain;

s6, cell Cy3 staining: and directly fluorescence labeling the fourth cell strain by using a second fluorescent dye Cy3 to obtain multiple fluorescence labeled living cells.

Based on the scheme, the invention can also be improved as follows:

further, each recombinant gene was prepared by a gene editing technique in S1.

Further, the eukaryotic expression vector is selected from one of pCAG series, and the liposome transfection reagent adopts Lipofectamine ^TM 3000。

Further, the specific steps in S3 are as follows: fully mixing a liposome transfection reagent, a first plasmid and a second plasmid, incubating for 5-30 min at room temperature, adding into a first cell strain, and incubating for 5-10 h in a carbon dioxide incubator at 35-38 ℃ to obtain a first cell strain expressing fluorescent protein mCerulean, mKate, tagBFP and mAmetrine; and fully mixing the liposome transfection reagent, the third plasmid and the fourth plasmid, adding the mixture into a second cell strain at room temperature for 5-30 min, and incubating the mixture in a carbon dioxide incubator at 35-38 ℃ for 5-10 h to obtain the second cell strain expressing LSSmCherry, mClover and EBFP 2.0.

And (2) placing the third target cells in a PBS buffer solution containing CFSE in S4, and incubating the third target cells in a carbon dioxide incubator at the temperature of between 35 and 38 ℃ for 10 to 30 minutes to obtain the third cell strain, wherein the concentration of the CFSE in the PBS buffer solution is between 2 and 10 mu mol/L.

Further, the trypsin digestion process of the first cell line, the second cell line and the third cell line in S5 includes: pancreatin is added into the cell strain for digestion, then the cell state is observed under a microscope, and after the cells are gradually shrunken and rounded, the pancreatin is immediately sucked out, and then a complete culture medium is added to suspend the cell strain.

Further, in S6, the fourth cell strain is placed in a PBS buffer solution containing Cy3, and incubated for 5-15 min in a carbon dioxide incubator at the temperature of 35-38 ℃ to obtain multiple fluorescence labeled living cells, wherein the concentration of Cy3 in the PBS buffer solution is 2-10 mu mol/L.

Further, the second fluorescent dye Cy3 is coupled with a lipophilic group, facilitating the binding of Cy3 to the cell membrane.

Based on the technical scheme of the invention, the method has the following beneficial effects:

(1) Nine fluorophores which can be used simultaneously and can be resolved are screened out, a subcellular structure marking strategy is designed and optimized, a specific two-photon selective excitation fluorescence microscopic imaging system is combined, and a direct fluorescence marking and fluorescent protein in-situ marking technology is combined, so that nine fluorescent markers can be simultaneously realized in a living cell sample, and at most nine target structures can be simultaneously observed in one-time imaging, and nine-color fluorescence microscopic imaging of living cells is realized.

Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Drawings

FIG. 1 shows the results of fluorescence microscopy of the living cell samples obtained in example 1.

Detailed Description

The following detailed description of embodiments of the invention is intended to be illustrative of the invention and is not to be taken as limiting the invention.

Experimental materials: the reagents and materials used in the examples and comparative examples of the present invention were all commercially available unless otherwise specified. Wherein, lipofectamine is adopted as liposome transfection reagent ^TM 3000 (ThermoFisher), the specific preparation method of the liposome transfection reagent is referred to the manual of lipo 3000; the blood-reducing culture medium is Opti-MEM ^TM (thermo fisher company); the eukaryotic expression vector used was pCAG-sleep. Fluorescent staining reagent CFSE (Sigma, 5 (6) -carboxydiacetic acid fluorescein succinimidyl ester), dissolved in DMSO, was prepared into CFSE stock solution at a concentration of 5 mmols; the fluorescence staining reagent Cy3 dye was Cy3-NHS-ester dye (Absin Co.) and dissolved in DMSO to prepare Cy3 stock at a concentration of 5mMolAnd (3) liquid.

The preparation method of the multiple fluorescent marked living cell sample based on the invention comprises the following steps:

s1, gene fusion: respectively connecting a dark blue fluorescent protein EBFP2.0 gene with a fibrin gene to form a recombinant EBFP2.0 gene, connecting a red fluorescent protein mKate gene with pyruvate dehydrogenase to form a recombinant mKate gene, connecting a blue fluorescent protein TagBFP gene with a histone gene to form a recombinant TagBFP gene, connecting a green fluorescent protein mAmetrine gene with a reticulin gene to form a recombinant mAmetrine gene, connecting a cyan fluorescent protein mCerulean gene with a beta-actin gene to form a recombinant mCerulean gene, and connecting a green fluorescent protein mClover3 gene with a sialyltransferase (SiT-15) gene to form a recombinant mClover3 gene;

s2, constructing plasmids: inserting the recombinant mCerulean gene and the recombinant mKate gene into a eukaryotic expression vector to construct a first plasmid for coexpression of cyan fluorescent protein mCerulean and red fluorescent protein mKate; inserting the recombinant tagBFP gene and the recombinant mAmetrine gene into a eukaryotic expression vector, and constructing a second plasmid (recorded as Tag-mA) for coexpression of the blue fluorescent protein tagBFP and the green fluorescent protein mAmetrine; inserting the LSSmCherry gene and the recombinant mClover3 gene into a eukaryotic expression vector to construct a third plasmid (denoted as LSS-Clo) for co-expressing the red fluorescent protein LSSmCherry and the green fluorescent protein mClover 3; the EBFP2.0 gene was inserted into a eukaryotic expression vector to construct a fourth plasmid for expressing the dark blue fluorescent protein EBFP 2.0.

Because the emission spectrum of the existing fluorophores is generally wider (the full width at half maximum of the spectrum is 40-50 nm), and the Stokes shift difference of the conventional fluorescent molecules is not large (generally 10-20 nm), the types of fluorophores for realizing the multiplex fluorescence specificity labeling are limited; meanwhile, in order to achieve as many fluorescent markers as possible in the same cell, it is common to select as many fusion of a plurality of fluorescent proteins as possible in one plasmid, but the larger the plasmid is, the more toxic the cell is, resulting in too low transfection efficiency of the cell, thereby easily causing apoptosis, and in order to express all the fluorescent proteins into the cell, the method of cotransfection (i.e., transfection of a plurality of plasmids in the same cell) is considered first, but because the loading capacity of the cell is limited and the plasmids are expressed in the cell, there is also a problem that some plasmids are preferentially expressed, the same cell cannot express too many plasmids introduced from the outside at the same time, and thus the number of fluorescent marker substances and organelles that can be simultaneously observed in the same cell is limited. The invention increases the kinds of simultaneously observable targets in a living cell sample to nine kinds through a series of experimental condition screening and experimental design, and specifically comprises the following steps:

Selection of fluorophores: firstly, nine fluorophores which can be imaged simultaneously and can be resolved are screened, and 3 fluorophores (EBFP 2.0, tagBFP, mCerulean; mAmetrine, mClover, mKate; LSSmCherry, CFSE and Cy 3) with far two-photon excitation peaks are respectively selected from the fluorophores with emission wavelengths of 450nm, 550nm and 650nm, wherein the nine fluorophores can realize fluorescence microscopic imaging of all marked targets through multichannel fluorescence signal detection and two-photon excitation wavelength tuning. As shown in table 1, the selected fluorophores included fluorescent proteins EBFP2.0, tagBFP, mCerulean, mAmetrine, mClover, mKate, and lssmchenry, and fluorescent dyes: CFSE and Cy3.

Labeling target selection: for fluorescent proteins, a protein fusion mode can be used for constructing corresponding plasmids, and a liposome transfection method is used for enabling gene fragments to enter cells so as to express fluorescence on specific proteins in the cells, so that subcellular structure markers are realized. By linking the dark blue fluorescent protein EBFP2.0 gene to the fibrin gene, EBFP2.0 can be expressed at the nucleolus; the blue fluorescent protein tagBFP gene is connected with a histone gene, so that the blue fluorescent protein tagBFP gene can be expressed at the cell nucleus; linking cyan fluorescent protein mCerulean with beta-actin gene to make it express on cell skeleton; the green fluorescent protein mAmetrine can be expressed in an endoplasmic reticulum by connecting the protein mAmetrine with a reticulin gene; the green fluorescent protein gene mClover3 can be expressed in the Golgi apparatus by linking it with sialyltransferase (SiT-15) gene; by linking red fluorescent protein mKate to pyruvate dehydrogenase (pyruvate dehydrogenase) gene, it can be expressed in mitochondria; expressing red fluorescent protein lssmchemry throughout the cell; in addition, fluorescent dye CFSE and Cy3 are selected for fluorescent labeling, CFSE can be used for cytoplasmic labeling, CFSE can easily penetrate through cell membranes, can be covalently bound with intracellular proteins in living cells, and release green fluorescence after hydrolysis, and in the process of cell division and proliferation, the fluorescence intensity of the fluorescent dye CFSE can be gradually decreased along with cell division, and the labeled fluorescence can be evenly distributed into two sub-generation cells, so that the fluorescence intensity is half of that of a parent cell, and according to the characteristic, the fluorescent dye CFSE can be used for detecting cell proliferation, and in the aspect of estimation of cell cycle, cy3-NHS-ester can be directly bound with the cell membranes and used for displaying the cell membrane state.

Table 1, each fluorophore and its single photon excitation wavelength, two photon excitation wavelength, fluorescence emission wavelength, and cell localization.

Underlined are fluorescent dye designations, others are fluorescent protein designations

Nine fluorophores which can be used simultaneously and can be resolved are screened out, a subcellular structure marking strategy is designed and optimized, a specific two-photon selective excitation fluorescence microscopic imaging system is combined, a direct fluorescence marking and fluorescent protein in-situ marking technology is combined, nine-fold fluorescence marking can be realized in a living cell sample at the same time, and therefore, at most nine target structures can be observed simultaneously in one-time imaging, and living cell nine-color fluorescence microscopic imaging is realized.

According to the preparation method of the multiple fluorescent labeling living cell sample, each recombinant gene is prepared in S1 through a gene editing technology.

Based on the preparation method of the multiple fluorescent labeling living cell sample, the eukaryotic expression vector is selected from one of pCAG series, such as pCAG-Flep, and the liposome transfection reagent adopts Lipofectamine ^TM 3000(ThermoFisher)。

The preparation method of the multiple fluorescent labeling living cell sample based on the embodiment of the invention comprises the following specific steps in S3: fully mixing a liposome transfection reagent, a first plasmid and a second plasmid, incubating for 5-30 min at room temperature, adding into a first cell strain, and incubating for 5-10 h in a carbon dioxide incubator at 35-38 ℃ to obtain a first cell strain expressing fluorescent protein mCerulean, mKate, tagBFP and mAmetrine; and fully mixing the liposome transfection reagent, the third plasmid and the fourth plasmid, adding the mixture into a second cell strain at room temperature for 5-30 min, and incubating the mixture in a carbon dioxide incubator at 35-38 ℃ for 5-10 h to obtain the second cell strain expressing LSSmCherry, mClover and EBFP 2.0.

According to the preparation method of the multiple fluorescent labeling living cell sample, in S4, the third target cells are placed in a PBS buffer solution containing CFSE, and incubated for 10-30 min in a carbon dioxide incubator at 35-38 ℃ to obtain the third cell strain, wherein the concentration of the CFSE in the PBS buffer solution is 2-10 mu mol/L.

According to the preparation method of the multiple fluorescent-labeled living cell sample, in S5, trypsin digestion processes of the first cell strain, the second cell strain and the third cell strain respectively comprise: pancreatin is added into the cell strain for digestion, then the cell state is observed under a microscope, and after the cells are gradually shrunken and rounded, the pancreatin is immediately sucked out, and then a complete culture medium is added to suspend the cell strain.

According to the preparation method of the multiple fluorescent marked living cell sample, provided by the embodiment of the invention, in S6, the fourth cell strain is placed in a PBS buffer solution containing Cy3, and is incubated for 5-15 min in a carbon dioxide incubator at the temperature of 35-38 ℃, so that multiple fluorescent marked living cells are obtained, and the concentration of Cy3 in the PBS buffer solution is 2-10 mu mol/L.

According to the preparation method of the multiple fluorescent labeling living cell sample, disclosed by the embodiment of the invention, the second fluorescent dye Cy3 is coupled with a lipophilic group, such as Cy3-NHS-ester, so that Cy3 can be conveniently combined with a cell membrane.

Example 1

The preparation method of the multiple fluorescent marked living cell sample comprises the following steps:

1. cell inoculation: heLa cells cultured in a cell bottle are digested, collected and centrifuged, after the supernatant is removed, the cells are resuspended into single cell suspension by using complete medium DMEM, 2-4X 104 cells are inoculated in each well of a 96-well plate, 3 wells are inoculated for standby, the serial numbers are A, B, C respectively, and the cells are cultured overnight.

2. Gene fusion: the method comprises the steps of adopting a gene editing technology, respectively connecting a dark blue fluorescent protein EBFP2.0 gene with a fibrin gene to form a recombinant EBFP2.0 gene, connecting a red fluorescent protein mKate gene with a pyruvate dehydrogenase gene to form a recombinant mKate gene, connecting a blue fluorescent protein TagBFP gene with a histone gene to form a recombinant TagBFP gene, connecting a green fluorescent protein mAmetrine gene with a reticulin gene to form a recombinant mAmetrine gene, connecting a cyan fluorescent protein mCerulean gene with a beta-actin gene to form a recombinant mCerulean gene, and connecting a green fluorescent protein mClover3 gene with a SiT-15 gene to form a recombinant mClover3 gene.

3. Constructing a plasmid: the recombinant mCerulean gene and the recombinant mKate gene are inserted into a eukaryotic expression vector pCAG-Flep to construct a first plasmid (marked as mC-mK) for coexpression of a cyan fluorescent protein mCerulean and a red fluorescent protein mKate, the recombinant TagBFP gene and the recombinant mAmetrine gene are inserted into a eukaryotic expression vector to construct a second plasmid (marked as Tag-mA) for coexpression of a blue fluorescent protein TagBFP and a green fluorescent protein mAmetrine, the LSSmCherry gene and the recombinant mClover3 gene are inserted into a eukaryotic expression vector to construct a third plasmid (marked as LSS-Clo) for coexpression of a red fluorescent protein LSSmCherry and a green fluorescent protein mClover3, and the EBFP2.0 gene is inserted into the eukaryotic expression vector to construct a fourth plasmid for expression of a deep blue fluorescent protein EBFP 2.0.

4. Cell transfection: the 96-well plate was removed from the incubator, the medium in both A/B wells was aspirated by a micropipette, and after washing with PBS once, the serum-reduced medium opti-MEM was added, followed by reference to liposome transfection reagent lipo3000 (Lipofectamine) ^TM 3000 2 tubes of liposome dilutions were prepared using a serum-reduced medium, numbered A, B, then equal amounts of mK-mC and Tag-mA plasmids were added to tube a, LSS-Clo plasmids and EBFP2.0 plasmids were added to tube B, liposomes and DNA were thoroughly mixed using a micropipette at 30 quenching in each of the separate tubes, after 15 minutes incubation at room temperature, DNA-liposome complexes in two A, B tubes were added to A, B two-well cells, respectively, after 6 hours incubation in a 37 ℃ carbon dioxide incubator, the medium in the A, B wells was aspirated, and the serum-reduced medium was replaced with complete medium.

5. Cell CFSE staining: the complete medium in the C hole of the 96-well plate is sucked out, PBS is used for cleaning residual serum, 1ml of PBS is taken in a centrifuge tube, 1 mu l of CFSE storage solution is added, the mixture is uniformly mixed to prepare 5 mu Mol of CFSE diluent, then 100 mu l of CFSE diluent is taken and added into the C hole, the complete medium is sucked out after the plate is placed in an incubator for incubation for 30min, PBS is used for flushing 2 times, and the complete medium is replaced, so that the cytoplasm can be observed to be marked with CFSE fluorescence under a mercury lamp.

6. Cell mixed inoculation: adding 200ul of complete culture medium into a hole in the middle of a 15mm confocal imaging small dish, putting the hole into an incubator for balancing, taking out the original culture medium in A, B, C holes, flushing the hole with a proper amount of PBS, adding a proper amount of pancreatin into each hole for digestion, observing the cell state under a microscope, immediately sucking out the pancreatin after the cells are gradually shrunken and rounded, adding 100ul of complete culture medium into each hole, suspending the cells in the hole, adding all 3 kinds of cells into a 1ml centrifuge tube, fully blowing and mixing, adding the fully mixed single cell suspension into the balanced confocal imaging small dish, putting the incubator, and culturing overnight in the incubator.

7. Cell Cy3 staining: the complete medium in the confocal imaging petri dish inoculated the previous day was aspirated, washed once with PBS, then 1ml of PBS was taken in a centrifuge tube, and 1. Mu.l of Cy3-NHS-ester stock solution was added, and mixed well to make 5. Mu.l of Cy3 diluent, 200. Mu.l of Cy3 diluent was added to the imaging petri dish, incubated at room temperature for 10min, after incubation was completed, cy3 diluent was completely aspirated, and then washed 3 times with PBS, at which time all cell membranes of cells were observed to have been labeled with Cy3 fluorescence under a mercury lamp, giving a nine-color fluorescence labeled viable cell sample.

Example 2

step one, cell inoculation: the B16 cells cultured in the cell flask are digested, collected and centrifuged, after the supernatant is removed, the cells are resuspended into single cell suspension by using a complete culture medium 1640, 2-4×104 cells are inoculated in each well of a 96-well plate, 3 wells are inoculated for standby, and the cells are respectively numbered A, B, C and cultured overnight.

Step two, gene fusion: the method comprises the steps of adopting a gene editing technology, respectively connecting a dark blue fluorescent protein EBFP2.0 gene with a fibrin gene to form a recombinant EBFP2.0 gene, connecting a red fluorescent protein mKate gene with a pyruvidehydrogenase gene to form a recombinant mKate gene, connecting a blue fluorescent protein TagBFP gene with a histone gene to form a recombinant TagBFP gene, connecting a green fluorescent protein mAmetrine gene with a reticulin gene to form a recombinant mAmetrine gene, connecting a cyan fluorescent protein mCerulean gene with a beta-actin gene to form a recombinant mCerulean gene, and connecting a green fluorescent protein mClover3 gene with a SiT-15 gene to form a recombinant mClover3 gene.

Step three, constructing plasmids: the recombinant mCerulean gene and the recombinant mKate gene are inserted into a eukaryotic expression vector pCAG-Flep to construct a first plasmid (marked as mC-mK) for coexpression of a cyan fluorescent protein mCerulean and a red fluorescent protein mKate, the recombinant TagBFP gene and the recombinant mAmetrine gene are inserted into a eukaryotic expression vector to construct a second plasmid (marked as Tag-mA) for coexpression of a blue fluorescent protein TagBFP and a green fluorescent protein mAmetrine, the LSSmCherry gene and the recombinant mClover3 gene are inserted into a eukaryotic expression vector to construct a third plasmid (marked as LSS-Clo) for coexpression of a red fluorescent protein LSSmCherry and a green fluorescent protein mClover3, and the EBFP2.0 gene is inserted into the eukaryotic expression vector to construct a fourth plasmid for expression of a deep blue fluorescent protein EBFP 2.0.

Step four, cell transfection: taking out the 96-well plate from the incubator, sucking out the culture medium in the two holes A/B by using a micropipette, washing the culture medium once by using PBS, adding an opti-MEM of a serum-reduced culture medium, preparing 2 tubes of liposome diluent by using the serum-reduced culture medium by referring to a using manual of a liposome transfection reagent lipo3000, numbering A, B, adding equivalent amount of mK-mC and Tag-mA plasmid into the tube A, adding LSS-Clo plasmid and EBFP2.0 plasmid into the tube B, quenching 30 by using the micropipette in each separate tube, fully mixing the liposome and DNA, incubating for 15 minutes at room temperature, respectively adding the DNA-liposome complex in the two tubes A, B into the cells in the two holes A, B, incubating for 6 hours in the carbon dioxide incubator at 37 ℃, sucking out the culture medium in the holes A, B, and replacing the serum-reduced culture medium by using a complete culture medium.

Step five, cell CFSE staining: the complete culture medium in the C hole of the 96-well plate is sucked out, PBS is used for cleaning residual serum, 1ml of PBS is taken to be placed in a centrifuge tube, CFSE storage solution is added, the mixture is uniformly mixed to prepare 2 mu Mol of CFSE diluent, then 100 mu l of CFSE diluent is taken to be added into the C hole, the complete culture medium is replaced by putting the well plate into an incubator for incubation for 20min, the complete culture medium is washed 2 times by PBS, and at the moment, the cytoplasm can be observed to be marked with CFSE fluorescence under a mercury lamp.

Step six, cell mixed inoculation: adding 200ul of complete culture medium into a hole in the middle of a 15mm confocal imaging small dish, putting the hole into an incubator for balancing, taking out the original culture medium in A, B, C holes, flushing the hole with a proper amount of PBS, adding a proper amount of pancreatin into each hole for digestion, observing the cell state under a microscope, immediately sucking out the pancreatin after the cells are gradually shrunken and rounded, adding 100ul of complete culture medium into each hole, suspending the cells in the hole, adding all 3 kinds of cells into a 1ml centrifuge tube, fully blowing and mixing, adding the fully mixed single cell suspension into the balanced confocal imaging small dish, putting the incubator, and culturing overnight in the incubator.

Step seven, cell Cy3 staining: the complete medium in the confocal imaging petri dish inoculated the previous day is sucked out, washed once by PBS, then 1ml of PBS is taken in a centrifuge tube, and Cy3-NHS-ester storage solution is added, and the mixture is uniformly mixed to prepare 2 mu Mol of Cy3 diluent, 200 mu l of Cy3 diluent is added in the imaging petri dish, incubated for 15min at room temperature, after the incubation is completed, the Cy3 diluent is completely sucked out, then washed 3 times by PBS, at the moment, the cell membranes of all cells can be observed to have been marked with Cy3 fluorescence under a mercury lamp, and a nine-color fluorescence marked living cell sample is obtained.

Example 3

step one, cell inoculation: heLa cells cultured in a cell bottle are digested, collected and centrifuged, after the supernatant is removed, the cells are resuspended into single cell suspension by using complete medium DMEM, 2-4X 104 cells are inoculated in each well of a 96-well plate, 3 wells are inoculated for standby, the serial numbers are A, B, C respectively, and the cells are cultured overnight.

Step four, cell transfection: taking out the 96-well plate from the incubator, sucking out the culture medium in the two holes A/B by using a micropipette, washing the culture medium once by using PBS, adding an opti-MEM of a serum-reduced culture medium, preparing 2 tubes of liposome diluent by using the serum-reduced culture medium by referring to a using manual of a liposome transfection reagent lipo3000, numbering A, B, adding equivalent amount of mK-mC and Tag-mA plasmid into the tube A, adding LSS-Clo plasmid and EBFP2.0 plasmid into the tube B, quenching 30 by using the micropipette in each separate tube, fully mixing the liposome and DNA, incubating for 15 minutes at room temperature, respectively adding the DNA-liposome complex in the two tubes A, B into the cells in the two holes A, B, incubating for 10 hours in the carbon dioxide incubator at 37 ℃, sucking out the culture medium in the holes A, B, and replacing the serum-reduced culture medium by using a complete culture medium.

Step five, cell CFSE staining: the complete culture medium in the C hole of the 96-well plate is sucked out, PBS is used for cleaning residual serum, 1ml of PBS is taken to be placed in a centrifuge tube, CFSE storage solution is added, the mixture is uniformly mixed to prepare 10 mu Mol of CFSE diluent, then 100 mu l of CFSE diluent is taken to be added into the C hole, the complete culture medium is replaced by completely sucking out the CFSE diluent after the well plate is placed in an incubator to be incubated for 10min, PBS is used for flushing for 2 times, and at the moment, the cytoplasm can be observed to be marked with CFSE fluorescence under a mercury lamp.

Step seven, cell Cy3 staining: the complete medium in the confocal imaging petri dish inoculated the previous day is sucked out, washed once by PBS, then 1ml of PBS is taken in a centrifuge tube, and Cy3-NHS-ester storage solution is added, and mixed evenly to prepare 10 mu Mol of Cy3 diluent, 200 mu l of Cy3 diluent is taken and added in the imaging petri dish, incubated for 5min at room temperature, after incubation, the Cy3 diluent is completely sucked out, then washed 3 times by PBS, at this time, the cell membranes of all cells can be observed to have been marked with Cy3 fluorescence under a mercury lamp, and a nine-color fluorescence marked living cell sample is obtained.

Comparative example 1

A method for preparing a fluorescent-labeled living cell sample, comprising the steps of:

step one, cell inoculation: heLa cells cultured in a cell bottle are digested, collected and centrifuged, after the supernatant is removed, the cells are resuspended into single cell suspension by using complete medium DMEM, 2-4×104 cells are inoculated in each well of a 96-well plate, 3 wells are inoculated for standby, the number is A, B, C, and the cells are cultured overnight.

Step three, inserting the recombinant mCerulean gene, the recombinant mKate gene, the recombinant TagBFP gene and the recombinant mAmetrine gene into the same eukaryotic expression vector (pCAG-Flep), constructing a first plasmid (marked as mC-mK-Tag-mAb) for coexpression of the cyan fluorescent protein mCerulean, the red fluorescent protein mKate, the blue fluorescent protein TagBFP and the green fluorescent protein mAmetrine, inserting the LSSmCherry gene and the recombinant mClover3 into the eukaryotic expression vector, constructing a second plasmid (marked as LSS-Clo) for coexpression of the fluorescent proteins LSSmCherry and the fluorescent protein mClover3, and constructing a plasmid (marked as EBFP 2.0) for independent expression of the EBFP2.0 gene.

Step four, cell transfection: taking out the 96-well plate from the incubator, sucking out the culture medium in the A, B two holes respectively by using a micropipette, washing the culture medium once by using PBS, adding a serum reduction culture medium, preparing 2 tubes of liposome diluent with reference to a use manual of lipo3000, numbering A, B, adding mC-mK-Tag-mA plasmid into the A tube, adding LSS-Clo plasmid and EBFP2.0 plasmid into the B tube, quenching 30 parts of the culture medium in each separate tube by using the micropipette, fully mixing the liposome and DNA, incubating the culture medium in the two A, B tubes of DNA-liposome complex into the A, B two holes respectively after 15 minutes at room temperature, sucking out the culture medium in the A, B holes after incubating in a 37 ℃ carbon dioxide incubator for 6 hours, and replacing the serum reduction culture medium with the complete culture medium.

Step five, cell CFSE staining: sucking out the complete culture medium in the C hole of the 96-well plate, and cleaning residual serum by using PBS; 1ml of PBS was placed in a centrifuge tube, 1. Mu.l of CFSE stock solution was added, and mixed well to prepare 5. Mu.l of CFSE diluent, then 100. Mu.l of CFSE diluent was added to the C-well, the well plate was placed in an incubator for 30min, and after incubation, the CFSE diluent was completely aspirated, and the complete medium was replaced by washing 2 times with PBS, at which time it was observed under a mercury lamp that the cytoplasm had been labeled with CFSE fluorescence.

Step six, cell mixed inoculation: adding 200ul of complete culture medium into a hole in the middle of a 15mm confocal imaging small dish, putting the hole into an incubator for balancing, taking out the original culture medium in A, B, C holes, flushing the hole with a proper amount of PBS, adding a proper amount of pancreatin into each hole for digestion, observing the cell state under a microscope, immediately sucking out the pancreatin after the cells are gradually shrunken and rounded, adding 100ul of complete culture medium into each hole, suspending the cells in the hole, adding all 3 kinds of cells into a 1ml centrifuge tube, fully blowing and mixing, adding the fully mixed single cell suspension into the balanced confocal imaging small dish, putting the incubator, and culturing the incubator overnight.

Step seven, cell Cy3 staining: the complete medium in the confocal imaging petri dish inoculated the previous day was aspirated, washed once with PBS, then 1ml of PBS was taken in a centrifuge tube, and 1. Mu.l of Cy3-NHS-ester stock solution was added, and mixed well to make 5. Mu.l of Cy3 diluent, 200. Mu.l of Cy3 diluent was added to the imaging petri dish, incubated at room temperature for 10min, after incubation was completed, cy3 diluent was completely aspirated, then washed 3 times with PBS, at which time all cell membranes of the cells were observed to have been labeled with Cy3 fluorescence under a mercury lamp, yielding a fluorescent-labeled viable cell sample.

Comparative example 2

Step three, constructing plasmids: inserting the recombinant mCerulean gene and the recombinant mKate gene into a eukaryotic expression vector (pCAG-Flep), constructing a first plasmid (marked as mC-mK) for coexpression of a cyan fluorescent protein mCerulean and a red fluorescent protein mKate, inserting the recombinant TagBFP gene and the recombinant mAmetrine gene into the eukaryotic expression vector, constructing a second plasmid (marked as Tag-mA) for coexpression of a blue fluorescent protein TagBFP and a green fluorescent protein mAmetrine, inserting the LSSmCherry gene, the recombinant mClover3 gene and the EBFP2.0 gene into the eukaryotic expression vector, and constructing a third plasmid (marked as LSS-Clo-EBFP 2.0) for coexpression of a red fluorescent protein LSSmCherry, a green fluorescent protein mClover3 and a deep blue fluorescent protein EBFP 2.0.

Step four, cell transfection: taking out the 96-well plate from the incubator, sucking out the culture medium in the A, B two wells respectively by using a micropipette, washing the culture medium once by using PBS, adding a serum reduction culture medium, preparing 2 tubes of liposome diluent by using the serum reduction culture medium with reference to a use manual of lipo3000, numbering A, B, adding equivalent amount of mK-mC plasmid and Tag-mA plasmid into the A tube, adding LSS-Clo-EBFP2.0 plasmid into the B tube, quenching 30 times in each separate tube by using the micropipette, fully mixing the liposome and DNA, incubating for 15 minutes at room temperature, adding the A, B two tubes of DNA-liposome complex into the A, B two wells respectively, incubating for 6 hours in a carbon dioxide incubator at 37 ℃, sucking out the culture medium in the A, B wells, and replacing the serum reduction culture medium by using the complete culture medium.

Step six, cell mixed inoculation: adding 200ul of complete culture medium into a hole in the middle of a 15mm confocal imaging small dish, putting the hole into an incubator for balancing, taking out the original culture medium in A, B, C holes, flushing the hole with a proper amount of PBS, adding a proper amount of pancreatin into each hole for digestion, observing the cell state under a microscope, immediately sucking out the pancreatin after the cells are gradually shrunken and rounded, adding 100ul of complete culture medium into each hole, suspending the cells in the hole, adding all 3 kinds of cells into one 1ml centrifuge tube, fully blowing and mixing, adding the fully mixed single cell suspension into the balanced confocal imaging small dish, putting the incubator, and culturing overnight in the incubator.

Comparative example 3

step one, cell inoculation: heLa cells cultured in the cell flask were digested, collected, centrifuged, and after the supernatant was removed, the cells were resuspended in single cell suspension using complete medium DMEM. 2-4×104 cells were seeded per well in 96-well plates, 3 wells were seeded for use, accession number A, C, and cultured overnight.

Step two, gene fusion: respectively connecting a dark blue fluorescent protein EBFP2.0 gene with a fibrin gene to form a recombinant EBFP2.0 gene, connecting a red fluorescent protein mKate gene with a pyruvate dehydrogenase gene to form a recombinant mKate gene, connecting a blue fluorescent protein TagBFP gene with a histone gene to form a recombinant TagBFP gene, connecting a green fluorescent protein mAmetrine gene with a reticulin gene to form a recombinant mAmetrine gene, connecting a cyan fluorescent protein mCerulean gene with a beta-actin gene to form a recombinant mCerulean gene, and connecting a green fluorescent protein mClover3 gene with a SiT-15 gene to form a recombinant mClover3 gene;

step three, constructing plasmids: inserting the recombinant mCerulean gene and the recombinant mKate gene into a eukaryotic expression vector pCAG-Flep to construct a first plasmid (marked as mC-mK) for coexpression of a cyan fluorescent protein mCerulean and a red fluorescent protein mKate; inserting the recombinant tagBFP gene and the recombinant mAmetrine gene into a eukaryotic expression vector pCAG-Flep to construct a second plasmid (marked as Tag-mAmetrine) for coexpression of the blue fluorescent protein tagBFP and the green fluorescent protein mAmetrine; inserting the LSSmCherry gene and the recombinant mClover3 gene into a eukaryotic expression vector to construct a third plasmid (denoted as LSS-Clo) for co-expressing the red fluorescent protein LSSmCherry and the green fluorescent protein mClover 3; the EBFP2.0 gene was inserted into a eukaryotic expression vector and a fourth plasmid (designated EBFP 2.0) was constructed for the expression of the dark blue fluorescent protein EBFP 2.0.

Step four, cell transfection: the 96-well plate was removed from the incubator, the medium in both a/B wells was aspirated using a micropipette, and after washing with PBS once, the serum-reduced medium was added, 2 tubes of liposome dilutions were prepared using the serum-reduced medium with reference to the use manual of lipo3000, no. a, then equal amounts of mK-mC plasmid, tag-mA plasmid, LSS-Clo plasmid and EBFP2.0 plasmid were added to the a tube, the liposomes and DNA were thoroughly mixed using a micropipette at 30 beats per separate tube, after incubation at room temperature for 15 minutes, the DNA-liposome complex in the a tube was added to the a well cells, after incubation in a carbon dioxide incubator at 37 ℃ for 6 hours, the medium in the a well was aspirated, and the serum-reduced medium was replaced with complete medium.

Step five, cell CFSE staining: the complete medium in the C hole of the 96-well plate is sucked out, PBS is used for cleaning residual serum, 1ml of PBS is taken in a centrifuge tube, 1 mu l of CFSE storage solution is added, the mixture is uniformly mixed to prepare 5 mu Mol of CFSE diluent, then 100 mu l of CFSE diluent is taken and added into the C hole, the complete medium is sucked out after the plate is placed in an incubator for incubation for 30min, PBS is used for flushing 2 times, and the complete medium is replaced, so that the cytoplasm can be observed to be marked with CFSE fluorescence under a mercury lamp.

Step six, cell mixed inoculation: adding 200ul of complete culture medium into a hole in the middle of a 15mm confocal imaging small dish, putting the hole into an incubator for balancing, taking out the original culture medium in A, C holes, flushing the hole with a proper amount of PBS, adding a proper amount of pancreatin into each hole for digestion, observing the state of the cells under a microscope, immediately sucking out the pancreatin after the cells are gradually shrunken and rounded, adding 100ul of complete culture medium into each hole, and suspending the cells in the holes; all 2 kinds of cell are added into a 1ml centrifuge tube, fully blown and mixed, fully mixed single cell suspension is added into a balanced confocal imaging small dish, and the mixture is placed into an incubator for overnight culture.

Step seven, cell Cy3 staining: the complete culture medium in the confocal imaging petri dish inoculated on the previous day is sucked out, PBS is used for cleaning once, 1ml of PBS is taken into a centrifuge tube, 1 μl of Cy3-NHS-ester storage solution is added, 5 μl of Cy3 diluent is prepared by uniformly mixing, 200 μl of Cy3 diluent is added into the imaging petri dish, incubation is carried out for 10min at room temperature, after incubation is completed, the Cy3 diluent is completely sucked out, then PBS is used for flushing 3 times, at the moment, the cell membranes of all cells can be observed to be marked with Cy3 fluorescence under a mercury lamp, and thus, the living cell marking is completed, and a fluorescent marked living cell sample is obtained.

Comparative example 4

step one, cell inoculation: heLa cells cultured in the cell flask were digested, collected, centrifuged, and after the supernatant was removed, the cells were resuspended in single cell suspension using complete medium DMEM. 2-4×104 cells were seeded per well in 96-well plates, 3 wells were seeded for use, accession number A, B, C, and cultured overnight.

step three, constructing plasmids: inserting the recombinant mCerulean gene, the recombinant mKate gene and the LSSmCherry gene into a eukaryotic expression vector pCAG-Flep to construct plasmids (marked as mC-mK-LSS) for coexpression of a cyan fluorescent protein mCerulean, a red fluorescent protein mKate and a red fluorescent protein LSSmCherry; the recombinant TagBFP gene, the recombinant mAmetrine gene, the recombinant mClover3 gene and the EBFP2.0 gene are inserted into eukaryotic expression vectors to construct second plasmids (marked as Tag-mA-Clo-EBFP 2.0) for co-expressing blue fluorescent protein TagBFP, green fluorescent protein mAmetrine, green fluorescent protein mClover3 and deep blue fluorescent protein EBFP 2.0.

Step four, cell transfection: taking out the 96-well plate from the incubator, sucking out the culture medium in the two holes A/B by using a micropipette, washing the culture medium once by using PBS, adding an opti-MEM as a serum reduction culture medium, preparing 2 tubes of liposome diluent by using the serum reduction culture medium with reference to a use manual of lipo3000, numbering A, B, adding the mC-mK-LSS plasmid in the tube A, adding the Tag-mA-Clo-EBFP2.0 plasmid in the tube B, quenching 30 by using the micropipette in each of the separate tubes, fully mixing the liposome and the DNA, incubating the DNA-liposome complex in the two tubes A, B into A, B two-hole cells respectively after 15 minutes at room temperature, and sucking out the culture medium in the holes A, B after incubating in a carbon dioxide incubator for 6 hours, and replacing the serum reduction culture medium by using a complete culture medium.

Step six, cell mixed inoculation: adding 200ul of complete culture medium into a hole in the middle of a 15mm confocal imaging small dish, putting the hole into an incubator for balancing, taking out the original culture medium in A, B hole cells, flushing the hole with a proper amount of PBS, adding a proper amount of pancreatin into each hole for digestion, observing the cell state under a microscope, immediately sucking out the pancreatin after the cells are gradually shrunken and rounded, adding 100ul of complete culture medium into each hole, suspending the cells in the hole, adding 2 kinds of cell cells into a 1ml centrifuge tube, fully blowing and mixing, adding the fully mixed single cell suspension into the balanced confocal imaging small dish, and putting the cell suspension into the incubator for overnight culture.

Comparative example 5

Step two, gene fusion: the dark blue fluorescent protein EBFP2.0 gene is respectively connected with a fibrin gene to form a recombinant EBFP2.0 gene, the red fluorescent protein mKate gene is connected with a pyruvate dehydrogenase gene to form a recombinant mKate gene, the blue fluorescent protein TagBFP gene is connected with a histone gene to form a recombinant TagBFP gene, the green fluorescent protein mAmetrine gene is connected with a reticulin gene to form a recombinant mAmetrine gene, the cyan fluorescent protein mCerulean gene is connected with a beta-actin gene to form a recombinant mCerulean gene, and the green fluorescent protein mClover3 gene is connected with a SiT-15 gene to form a recombinant mClover3 gene.

Step three, constructing plasmids: inserting the recombinant mCerulean gene and the recombinant mKate gene into a eukaryotic expression vector pCAG-Flep to construct a first plasmid (marked as mC-mK) for coexpression of a cyan fluorescent protein mCerulean and a red fluorescent protein mKate; inserting the recombinant tagBFP gene and the recombinant mAmetrine gene into a eukaryotic expression vector, and constructing a second plasmid (recorded as Tag-mA) for coexpression of the blue fluorescent protein tagBFP and the green fluorescent protein mAmetrine; inserting the LSSmCherry gene and the recombinant mClover3 gene into a eukaryotic expression vector to construct a third plasmid (denoted as LSS-Clo) for co-expressing the red fluorescent protein LSSmCherry and the green fluorescent protein mClover 3; the EBFP2.0 gene was inserted into a eukaryotic expression vector and a fourth plasmid (designated EBFP 2.0) was constructed for the expression of the dark blue fluorescent protein EBFP 2.0.

Step four, cell transfection: taking out the 96-well plate from the incubator, sucking out the culture medium in the A, B two wells by using a micropipette, washing the culture medium once by using PBS, adding opti-MEM, preparing 2 tubes of liposome diluent by using a serum reduction culture medium by referring to a manual of lipo3000, numbering A, B, adding equivalent amount of mK-mC plasmid and Tag-mA plasmid into the A tube, adding LSS-Clo plasmid and EBFP2.0 plasmid into the B tube, quenching 30 times in each separate tube by using the micropipette, fully mixing the liposome and DNA, incubating for 5 minutes at room temperature, respectively adding the A, B two tubes of DNA-liposome complex into A, B two wells of cells, incubating for 6 hours in a carbon dioxide incubator at 37 ℃, sucking out the culture medium in A, B, and replacing the serum reduction culture medium by using the complete culture medium.

Step five, cell CFSE staining: sucking out the complete culture medium in the C hole of the 96-well plate, cleaning residual serum by using PBS, taking 1ml of PBS into a centrifuge tube, adding 1 μl of CFSE storage solution, uniformly mixing to prepare 5 μl of CFSE diluent, and then taking 100 μl of CFSE diluent and adding into the C hole; placing the pore plate into an incubator for incubation for 30min, and completely sucking out the CFSE diluent; the complete medium was replaced by 2 washes with PBS, at which point the cytoplasm was observed to have been labeled with CFSE fluorescence under a mercury lamp.

Step six, staining cells by Cy 3: the complete medium in the well C of the 96-well plate was aspirated, washed once with PBS, then 1ml of PBS was taken in a centrifuge tube, and 1. Mu.l of Cy3-NHS-ester stock solution was added, and mixed well to prepare 5. Mu.l of Cy3 dilution, 200. Mu.l of Cy3 dilution was added to an imaging cuvette, incubated at room temperature for 10min, after incubation was completed, the Cy3 dilution was completely aspirated, and then washed 3 times with PBS, at which time it was possible to observe that the cell membranes of all cells had been labeled with Cy3 fluorescence under a mercury lamp.

Step seven, cell mixed inoculation: taking out the original culture medium in A, B, C-hole cells, washing the cells with a proper amount of PBS, adding a proper amount of pancreatin into each hole for digestion, observing the cell state under a microscope, immediately sucking out the pancreatin after the cells are gradually shrunken and rounded, adding 100ul of complete culture medium into each hole, suspending the cells in the holes, adding all 3 kinds of cell cells into a 1ml centrifuge tube, fully blowing and mixing, adding the completely mixed single cell suspension into a balanced confocal imaging cuvette, placing the cuvette into an incubator, and culturing overnight in the incubator, thus completing the labeling of living cells and obtaining a fluorescent labeled living cell sample.

Two-photon excitation fluorescence microscopy images were respectively performed on the living cell samples obtained in examples, comparative example 1, comparative example 2, comparative example 3, comparative example 4, and multi-example 5. The results of two-photon excitation fluorescence microscopy of the nine-color fluorescence labeled living cell sample in example 1 are shown in fig. 1, and in combination with a specific two-photon excitation wavelength, nine types of target structures (nucleoli, nucleus, β -actin, cytoplasm, endoplasmic reticulum, golgi apparatus, whole cell structure, cell membrane, mitochondria) can be observed in cells, respectively, and nine-color fluorescence labeling of living cells is achieved, and nine-color fluorescence microscopy of living cells can be performed. As a result of two-photon excitation fluorescence microscopic imaging of the fluorescent-labeled living cell sample obtained in comparative example 1, it was found that the four fluorescent protein genes of mCerulean, mKate, tagBFP and mamelline were directly constructed in the same plasmid, and less cells were transfected to express fluorescence, and fluorescence of mCerulean, mKate, LSSmCherry, mClover, EBFP 2.0.0, CFSE and Cy3 could not be observed in the imaging experiment. Two-photon excitation fluorescence microscopy was performed on the fluorescence-labeled viable cell samples obtained in comparative example 2, and as a result, it was found that three fluorescent protein genes LSSmCherry, mClover and EBFP2.0 were constructed in the same plasmid, and less cells were transfected to express fluorescence, fluorescence of mCerulean, mKate, tagBFP, mAmetrine, LSSmCherry, mClover, CFSE and Cy3 could be observed in the imaging experiment, and fluorescence from EBFP2.0 could not be observed. As a result of two-photon excitation fluorescence microscopic imaging of the fluorescent-labeled living cell sample obtained in comparative example 3, it was found that four kinds of plasmids (mC-mK, tag-mA, LSS-Clo, EBFP 2.0) were mixed and simultaneously transfected into cells, and only four kinds of fluorescent proteins mCerulean, mKate, tagBFP and mAmetrine were smoothly localized in the designed cell structure after transfection, and none of the other three kinds of fluorescent proteins (LSSmCherry, mClover and EBFP 2.0) was observed. Two-photon excitation fluorescence microscopy of the fluorescently labeled viable cell samples obtained in comparative example 4 revealed that mCerulean, mKate and lssmchemry three fluorescent proteins were constructed on the same plasmid, tagBFP, mAmetrine, mClover and EBFP2.0 four fluorescent proteins were constructed on the same plasmid, few cells expressing fluorescence after transfection, and no fluorescence was observed for both LSSmCherry, mClover and EBFP 2.0. As a result of two-photon excitation fluorescence microscopic imaging of the fluorescent-labeled living cell sample obtained in comparative example 5, it was found that Cy3 was very unstable in association with cell membrane, and Cy3 was metabolized into the medium by cells after several hours of staining, and finally, significant Cy3 fluorescence could not be observed.

The invention is based on a series of experiments, and found that mCerulean and mKate are constructed on the same plasmid (marked as mC-mK), tagBFP and mAmetrine are constructed on the same plasmid (marked as Tag-mA), LSSmCherry and mClover3 are constructed on the same plasmid (marked as LSS-Clo), EBFP2.0 is independently constructed into plasmids, and all four constructed plasmids can express fluorescence at specific protein positions during transfection, and have higher transfection efficiency, then mC-mK plasmids and Tag-mA plasmids are co-transfected, and seven kinds of fluorescence can be observed in cells and the cells expressing fluorescent proteins are more when LSS-Clo plasmids and EBFP2.0 plasmids are co-transfected; cy3 is very unstable in combination with cell membrane and is metabolized into the culture medium by cells after several hours of staining, so Cy3 cell membrane labeling is performed before observation, namely transfection is performed first, then cytoplasm and cell membrane staining is performed, and finally microscopic observation is performed. Based on the preparation method of the multiple fluorescent labeling living cell sample, which combines the direct fluorescent labeling and fluorescent protein in-situ labeling technology, the nine-fold fluorescent labeling can be realized in the living cell sample at the same time, and at most nine target structures can be observed at the same time in one-time imaging, so that at most nine-color living cell fluorescent microscopic imaging can be realized.

Although embodiments of the present invention have been described in detail above, one of ordinary skill in the art will appreciate that: many changes, modifications, substitutions and variations may be made to the embodiments without departing from the spirit and principles of the invention, the scope of which is defined by the claims and their equivalents.

Claims

1. A method for preparing a multiple fluorescent-labeled living cell sample, comprising the steps of:

s1, gene fusion: respectively connecting a dark blue fluorescent protein EBFP2.0 gene with a fibrin gene to form a recombinant EBFP2.0 gene, connecting a red fluorescent protein mKate gene with a pyruvate dehydrogenase gene to form a recombinant mKate gene, connecting a blue fluorescent protein TagBFP gene with a histone gene to form a recombinant TagBFP gene, connecting a green fluorescent protein mAmetrine gene with a reticulin gene to form a recombinant mAmetrine gene, connecting a cyan fluorescent protein mCerulean gene with a beta-actin gene to form a recombinant mCerulean gene, and connecting a green fluorescent protein mClover3 gene with a sialyltransferase gene to form a recombinant mClover3 gene;

s2, constructing plasmids: inserting the recombinant mCerulean gene and the recombinant mKate gene into a eukaryotic expression vector to construct a first plasmid for coexpression of cyan fluorescent protein mCerulean and red fluorescent protein mKate; inserting the recombinant tagBFP gene and the recombinant mAmetrine gene into a eukaryotic expression vector to construct a second plasmid for co-expressing the blue fluorescent protein tagBFP and the green fluorescent protein mAmetrine; inserting the LSSmCherry gene and the recombinant mClover3 gene into a eukaryotic expression vector to construct a third plasmid for coexpression of the red fluorescent protein LSSmCherry and the green fluorescent protein mClover 3; inserting the EBFP2.0 gene into a eukaryotic expression vector to construct a fourth plasmid for expressing the dark blue fluorescent protein EBFP 2.0;

s6, cell Cy3 staining: and directly performing fluorescent labeling on the fourth cell strain by using a second fluorescent dye Cy3 to obtain multiple fluorescent labeled living cells, wherein the second fluorescent dye Cy3 is Cy3-NHS-ester.

2. The method for preparing a multiple fluorescent-labeled living cell sample according to claim 1, wherein each recombinant gene is prepared by a gene editing technique in S1.

3. The method for preparing a multiple fluorescent-labeled living cell sample according to claim 1, wherein the eukaryotic expression vector is selected from one of the pCAG series, and the liposome transfection reagent is Lipofectamine ™ 3000.

4. The method for preparing a multiple fluorescent-labeled living cell sample according to claim 1, wherein the specific steps in S3 are as follows: fully mixing a liposome transfection reagent, a first plasmid and a second plasmid, incubating for 5-30 min at room temperature, adding into a first cell strain, and incubating for 5-10 h in a carbon dioxide incubator at 35-38 ℃ to obtain a first cell strain expressing fluorescent protein mCerulean, mKate, tagBFP and mAmetrine; and fully mixing the liposome transfection reagent, the third plasmid and the fourth plasmid, adding the mixture into a second cell strain at room temperature for 5-30 min, and incubating the mixture in a carbon dioxide incubator at 35-38 ℃ for 5-10 h to obtain the second cell strain expressing LSSmCherry, mClover and EBFP 2.0.

5. The method for preparing a multi-fluorescence-labeled living cell sample according to claim 1, wherein the third target cell is placed in a PBS buffer containing CFSE in S4, and incubated in a carbon dioxide incubator at 35-38 ℃ for 10-30 min, so as to obtain the third cell strain, and the concentration of CFSE in the PBS buffer is 2-10 mu mol/L.

6. The method of claim 1, wherein the trypsin digestion of the first, second and third cell lines in S5 comprises: pancreatin is added into the cell strain for digestion, then the cell state is observed under a microscope, and after the cells are gradually shrunken and rounded, the pancreatin is immediately sucked out, and then a complete culture medium is added to suspend the cell strain.

7. The method for preparing a multi-fluorescence-labeled living cell sample according to claim 1, wherein the fourth cell line is placed in a PBS buffer containing Cy3 in S6, and incubated in a carbon dioxide incubator at 35-38 ℃ for 5-15 min, thereby obtaining multi-fluorescence-labeled living cells, and the concentration of Cy3 in the PBS buffer is 2-10 μmol/L.