A fluorescent labeling method for real-time tracking of intracellular microsporidia proliferation and development
By using lipophilic carbon cyanine fluorescent dyes DiI or DiD to stain and label microsporidium, the problem of inability to track microsporidium proliferation and development in the prior art is solved, and live cell marking and real-time tracer of microsporidium at each development stage is achieved.
Patent Information
- Application Number
- CN202411745668.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-02
- Publication Date
- 2025-03-04
- Estimated Expiration
- 2044-12-02
AI Technical Summary
The prior art cannot track the proliferation and development process of microsporidium in host cells in real time, and cannot label the various development stages of microsporidium at the same time.
The mature spores of microsporidium were stained with lipophilic carbon cyanine fluorescent dye DiI or DiD, and the stained spores were inoculated into the host cells. The distribution of the dye was observed within 5 hours and 120 hours by laser confocal microscopy to track the proliferation and development of microsporidium in real time.
The live cell marking and real-time tracer of microsporidium and its various developmental stages have been achieved, and the problem of the inability to trace the proliferation and development of microsporidium in the prior art is solved, and the gap in the intracellular proliferation and development method based on live cells is filled.
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Figure CN119223711B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of fluorescent staining, and in particular relates to a fluorescent labeling method for real-time tracing of proliferation and development of intracellular microsporidia. Background Art
[0002] Microsporidia are a class of obligate intracellular parasites. About 220 genera and more than 1,700 species have been reported worldwide, and new species are reported every year. Microsporidia have a very wide host range and distribution, and can infect almost all invertebrates and vertebrates, such as humans, rabbits, silkworms, bees and crabs. Some microsporidia are important pathogens of humans, livestock, aquaculture, and economic insects such as silkworms and bees, causing serious losses to human health and economic development, but there is a lack of effective means to prevent and control microsporidia. As an intracellular pathogen, the lack of understanding of the mechanism of mature microsporidian spores infecting host cells and their subsequent intracellular proliferation and development and adaptation to intracellular survival is an important bottleneck hindering the development of microsporidian disease prevention and control measures. In the study of microsporidian cell biology and its interaction with the host, marking the cytosolic and intracellular proliferation and development of microsporidia is of great significance to revealing the intracellular adaptive survival mechanism of microsporidia. Currently, due to the lack of genetic manipulation technology for microsporidia, the use of transgenic technology to drive the expression of fluorescent marker genes such as GFP using specific strong promoters to obtain stable fluorescent tracing microsporidia strains is still an unexplored field.
[0003] At present, the commonly used methods for labeling intracellular microsporidia include immunological methods and fluorescent staining methods. Immunological methods are often based on the specific binding of antigens and antibodies. The immunological method first requires the in vitro expression and purification of the specific proteins of the known microsporidian schizonts or mature spores, followed by the preparation of polyclonal antibodies, and then combined with fluorescent secondary antibodies to achieve the localization of the microsporidian schizonts or mature spores. The fluorescent staining method mainly stains the chitin of the microsporidian spore wall, so the fluorescent staining method is also limited to labeling sporozoites and mature spores. In addition, whether it is an immunological method or a fluorescent staining method, the cells must first be fixed and permeabilized before the antibodies or dyes can enter the cells for labeling. Therefore, these two methods cannot simultaneously label all developmental stages of microsporidia, nor can they label live microsporidian cells, and thus cannot track the proliferation and development of microsporidia in real time. Summary of the invention
[0004] In order to solve the problem that the existing intracellular microsporidia labeling method cannot track the proliferation and development of microsporidia in real time, the present invention provides a fluorescent labeling method for real-time tracking of the proliferation and development of intracellular microsporidia. To achieve the above purpose, the present invention adopts the following technical solution.
[0005] The present invention provides a fluorescent labeling method for real-time tracing of the proliferation and development of intracellular microsporidia, comprising the following steps:
[0006] Collect mature spores of microsporidia.
[0007] The mature spores of the microsporidia are stained with a staining solution prepared with a lipophilic carbocyanine dye to obtain the stained mature spores of the microsporidia.
[0008] The stained mature spores of microsporidia are inoculated into the host cells being cultured for infection to obtain living cell samples.
[0009] The living cell samples were observed using a laser confocal microscope 5 to 120 hours after infection.
[0010] The lipophilic carbocyanine dye includes any one of DiI and DiD.
[0011] In the prior art, methods for labeling intracellular microsporidia mainly include immunological methods and fluorescent staining methods. Among them, the immunological method is usually to express the housekeeping gene or spore wall protein of the microsporidia in vitro, then immunize the animal to obtain the corresponding antibody, and use it in combination with a fluorescently labeled secondary antibody to finally achieve the labeling of the intracellular microsporidia schizonts or mature spores. The fluorescent staining method mainly labels the chitin of the microsporidia. Therefore, the fluorescent staining method can only label sporozoites and mature spores. In addition, whether it is an immunological method or a fluorescent staining method, the cells must be fixed and permeabilized first before the antibodies or dyes can enter the cells for labeling. Therefore, the existing immunological methods and fluorescent staining methods have the technical defects of being unable to synchronously label or trace the various developmental stages of microsporidia in real time, and unable to label living cells of microsporidia.
[0012] Based on solving the above technical defects, the present invention provides a fluorescent labeling method based on living cells, which can simultaneously label various proliferation stages of microsporidia and can track the proliferation and development of intracellular microsporidia in real time. DiI or DiD used in the present invention is a lipophilic carbocyanine fluorescent dye that can bind to lipid molecules in the cell membrane and embed into the cell membrane of living cells, and can remain on the cell membrane for a long time and pass to daughter cells with cell division. Therefore, the present invention can achieve the live cell labeling and real-time tracking of microsporidia and their various developmental stages. This method effectively solves the problem that the proliferation and development of microsporidia cannot be tracked in real time in the prior art, and fills the gap in the method of continuous tracking of intracellular proliferation and development of microsporidia based on living cells.
[0013] Preferably, the method for obtaining the stained mature spores of microsporidia comprises the following steps:
[0014] After the mature spores of the microsporidia are centrifuged, the spore precipitate is collected, and the spore precipitate is resuspended by adding the dyeing solution prepared by the lipophilic carbocyanine dye, and then kept at room temperature for 10 minutes; centrifuged again, the spore precipitate is collected and resuspended and washed with a PBS solution to obtain the stained mature spores of the microsporidia. At this time, the membrane structure of the mature spores of the microsporidia has been marked by the lipophilic carbocyanine dye to dynamically track the subsequent microsporidia infection of host cells and the proliferation and development process in the host cells.
[0015] Preferably, the centrifugation condition is 8000 g for 5 min. Centrifugation at 8000 g for 5 min can better collect the spore precipitate.
[0016] DiI and DiD are lipophilic fluorescent dyes that can be used to stain cell membranes and other lipid-soluble biological structures, and are used as long-term tracers for living cells or tissues. The two dyes have different excitation wavelengths, showing orange and red fluorescence colors respectively, and can be flexibly selected when combined with other dyes.
[0017] Preferably, the dyeing time is 10 minutes. The lipophilic carbocyanine dye has a good tracing effect on the proliferation and development of microsporidia when the dyeing time is 10 minutes.
[0018] Preferably, the final concentration of the dye solution prepared by the lipophilic carbocyanine dye is 5 μM. The lipophilic carbocyanine dye has a good tracing effect on the proliferation and development of microsporidia when the final concentration is 5 μM.
[0019] Preferably, the concentration of the PBS solution is 0.01 M and the pH is 7.3.
[0020] Preferably, the method of inoculating the stained mature spores of microsporidia into the host cells being cultured comprises the following steps:
[0021] When the host cells being cultured reach a confluence of 80%, the stained mature spores of the microsporidia are inoculated into the host cells being cultured for co-culture.
[0022] The co-culture conditions are 37°C, 5% CO2, and 95% humidity, which are the optimal growth conditions for rabbit kidney cells.
[0023] Preferably, within 5 to 120 hours after infection, the lipophilic carbocyanine dye has a good tracing effect on the proliferation and development of microsporidia.
[0024] Preferably, the host cell is a rabbit kidney cell. In a preferred embodiment of the present invention, rabbit kidney cells, the host cells of Myospora swimming crab, are used as research objects, and the present invention is also applicable to host cells of other microsporidia.
[0025] Preferably, the microsporidia are Myospora swimming crab. In a preferred embodiment of the present invention, Myospora swimming crab is used as the research object, and the present invention is also applicable to other species of microsporidia.
[0026] Compared with the prior art, the present invention has the following beneficial effects:
[0027] 1. A fluorescent labeling method for real-time tracing of intracellular microsporidia proliferation and development provided by the present invention solves the problem of tracing microsporidia based on transgenic technology, fills the gap of real-time tracing of microsporidia at different developmental stages in cells, and greatly reduces scientific research costs and technical requirements; it replaces the complex method of marking microsporidia at different proliferation periods based on two-color or multi-color fluorescent staining steps of cell fixation and permeability, is easy to operate, has simple steps, does not require professional technicians, and greatly shortens the experimental time. The fluorescent labeling method for real-time tracing of intracellular microsporidia proliferation and development provided by the present invention does not need to be performed on the basis of cell fixation and permeability, does not destroy the cell structure and antigenic epitopes, can accurately reflect the real situation in living cells, effectively solves the problem that the proliferation and development of microsporidia cannot be traced in real time in the prior art, and fills the gap of the method for continuous tracing of intracellular proliferation and development of microsporidia based on living cells.
[0028] 2. Currently, there is a lack of methods for simultaneously labeling each developmental stage of microsporidia and methods for continuously tracing their intracellular proliferation and development based on living cells; existing fluorescent labeling methods for intracellular microsporidia proliferation and development require more than one method or dye, which causes inconvenience to the research, and all need to be carried out on the basis of cell fixation and permeabilization, which may be difficult to accurately reflect the real situation in living cells. The fluorescent labeling method for real-time tracing of intracellular microsporidia proliferation and development provided by the present invention can effectively solve the above problems.
[0029] 3. The fluorescent labeling method for real-time tracing of intracellular microsporidia proliferation and development provided by the present invention is easy to combine with multicolor fluorescent labeling to study the real-time "ecological niche" of microsporidia in cells and real-time interaction with host organelles; the carbocyanine dye family in the fluorescent labeling method for real-time tracing of intracellular microsporidia proliferation and development of the present invention includes two with different spectra: DiI and DiD, with maximum excitation wavelengths of 549nm and 644nm, respectively, and a narrow chromatographic range, which can be flexibly selected when used in combination with other dyes, and has great application potential.
[0030] 4. The fluorescent labeling method for real-time tracing of intracellular microsporidia proliferation and development provided by the present invention also has the following advantages:
[0031] (1) The dye solution prepared with lipophilic carbocyanine dyes can dye the mature spores of microsporidia without affecting their infectious activity.
[0032] (2) The staining solution prepared with lipophilic carbocyanine dyes can mark the various developmental stages of intracellular development of microsporidia, including schizogony and spore reproduction.
[0033] (3) Laser confocal microscopy can be used to observe the intracellular development stages of microsporidia at different time points after infection in real time. BRIEF DESCRIPTION OF THE DRAWINGS
[0034] Figure 1 The image is of the mature spores of Myospora swimming crab after DiI staining in the present invention during the period of 5 hours to 120 hours of infecting rabbit kidney cells; wherein, Figure 1 A in the figure is an image of mature spores of Myospora swimmingii stained with DiI after infecting rabbit kidney cells for 5 hours, from left to right are DiI channel and bright field overlay image, DiI channel image, and bright field image; Figure 1 B in the figure is an image of mature spores of Myospora swimming crab stained with DiI after infecting rabbit kidney cells for 1 day, from left to right are DiI channel and bright field overlay image, DiI channel image, and bright field image; Figure 1 C in the figure is an image of mature spores of Myospora swimmingii stained with DiI after infecting rabbit kidney cells for 2 days, from left to right are DiI channel and bright field overlay image, DiI channel image, and bright field image; Figure 1 D in the figure is an image of mature spores of Myosodium swimming crab after DiI staining, which infected rabbit kidney cells for 120 hours. From left to right are DiI channel and bright field overlay image, DiI channel image, and bright field image.
[0035] Figure 2 This is an image of mature spores of Myospora swimming crab after DiD staining in the present invention when infecting rabbit kidney cells for 1 day; wherein, Figure 2 A in the figure is the superimposed image of DiD channel and bright field; Figure 2 B in the figure is the DiD channel image; Figure 2 C in the figure is a bright field image. DETAILED DESCRIPTION
[0036] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.
[0037] The preparation method of the DiI staining solution used in Example 1 is as follows: 10 mg of DiI powder is dissolved in 2 mL of DMSO solution to prepare a first staining solution stock solution with a concentration of 5 mM, and the stock solution is wrapped in tin foil and stored at -20°C in the dark. Before staining, the first staining solution stock solution is diluted 1000 times in PBS solution to prepare a DiI staining solution with a final concentration of 5 μM.
[0038] Among them, DiI powder was purchased from Biyuntian Biotechnology Co., Ltd.; PBS solution was purchased from Solebow Technology Co., Ltd., with a concentration of 0.01 M and a pH of 7.3.
[0039] The preparation method of the DiD staining solution used in Example 2 is as follows: 10 mg of DiD powder is dissolved in 2 mL of DMSO solution to prepare a second staining solution mother solution with a concentration of 5 mM, and the second staining solution mother solution is wrapped in tin foil and stored at -20°C in the dark. Before staining, the second staining solution mother solution is diluted 1000 times in PBS solution to prepare a DiD staining solution with a final concentration of 5 μM.
[0040] Among them, DiD powder was purchased from Biyuntian Biotechnology Co., Ltd.; PBS solution was purchased from Solebow Technology Co., Ltd., with a concentration of 0.01 M and a pH of 7.3.
[0041] The experimental materials in Examples 1 and 2 are all swimming crab myospora and rabbit kidney cells, both of which are from the College of Marine Science and Engineering, Qingdao Agricultural University.
[0042] Example 1
[0043] A fluorescent labeling method for real-time tracing of intracellular microsporidia proliferation and development comprises the following steps:
[0044] Collect 50 µL of 1х10 11 The free mature spores of Myosporea of swimming crab at 100 / mL were centrifuged at 8000g for 5min. After centrifugation, the lower spore sediment was collected and resuspended with 1mL PBS solution to wash the lower spore sediment, centrifuged again, the supernatant was removed, and 1mL DiI staining solution with a final concentration of 5μM was added to a 1.5mL centrifuge tube and resuspended, kept at room temperature for 10min, centrifuged at 8000g for 5min, the supernatant was removed, the lower sediment was collected and resuspended with 1mL PBS solution to wash the sediment twice. After the sediment was resuspended with PBS solution again, it was counted under a wide-field fluorescence microscope using a hemocytometer to obtain the stained mature spores of Myosporea of swimming crab.
[0045] 1х10 5 Rabbit kidney cells were inoculated into laser confocal cell culture dishes containing 1 mL of complete culture medium and cultured at 37°C, 5% CO2, and 95% humidity until the confluence reached 80%. The stained mature spores of Myospora cypridina were plated at 10 8The concentration of spores was inoculated into the above culture dish. The old culture medium was discarded every two days and replaced with new complete culture medium. At 5 hours, 24 hours, 48 hours and 120 hours after the stained spores were infected, the monolayer cells in the confocal culture dish were carefully washed twice with PBS solution and new complete culture medium was added to obtain living cell samples. The obtained living cell samples were placed under a laser confocal microscope, and the excitation light wavelength was selected to be 561nm to observe the living cell samples.
[0046] The complete culture medium was high-glucose DMEM medium supplemented with 10% V / V fetal bovine serum, 100 U / mL penicillin, and 100 µg / mL streptomycin.
[0047] The live cell samples are rabbit kidney cells infected with mature spores of Mycocystis cynomolgus after staining.
[0048] The results of the stained mature spores of Myospora spores infecting rabbit kidney cells are shown in the figure. Figure 1 Shown are images of stained mature spores of Mycocystis spp. in rabbit kidney cells from 5 to 120 hours of infection.
[0049] Depend on Figure 1 The results can observe the process of mature spores or cytoplasm of swimming crab myospores from never entering rabbit kidney cells, entering rabbit kidney cells to proliferating in rabbit kidney cells, and the imaging detection effect is excellent.
[0050] The above results show that DiI carbocyanine dyes can track in real time the process of mature spores of free swimming crab myospores from infecting rabbit kidney cells to proliferating and developing in rabbit kidney cells.
[0051] Example 2
[0052] A fluorescent labeling method for real-time tracing of intracellular microsporidia proliferation and development comprises the following steps:
[0053] Collect 50 µL of 1х10 11 The mature spores of free swimming crab myospores were centrifuged at 8000g for 5min. After centrifugation, the lower spore precipitate was collected and resuspended with 1mL PBS solution to wash the lower spore precipitate, centrifuged again, and the supernatant was removed. After resuspending in 1mL DiD staining solution with a final concentration of 5μM in a 1.5mL centrifuge tube, the mixture was kept at room temperature for 10min, centrifuged at 8000g for 5min, the supernatant was removed, the lower precipitate was collected and resuspended with 1mL PBS solution to wash the precipitate twice. After resuspending in PBS solution again, the spores were counted under a wide-field fluorescence microscope using a hemocytometer to obtain the stained mature spores of swimming crab myospores.
[0054] 1х10 5Rabbit kidney cells were inoculated into laser confocal cell culture dishes containing 1 mL of complete culture medium and cultured at 37°C, 5% CO2, and 95% humidity until the confluence reached 80%. 8 The concentration of spores was inoculated into the above culture dish. The old culture medium was discarded every two days and replaced with new complete culture medium. One day after the stained spores were infected, they were carefully washed twice with PBS solution and new complete culture medium was added to obtain live cell samples. The obtained live cell samples were placed under a laser confocal microscope, and the excitation light wavelength was selected to be 640nm to observe the live cell samples in the confocal culture dish.
[0055] The complete culture medium is composed of high-glucose DMEM medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 µg / mL streptomycin at a V / V ratio.
[0056] The live cell samples are rabbit kidney cells infected with mature spores of Mycocystis cynomolgus after staining.
[0057] The results of the stained mature spores of Myospora spores infecting rabbit kidney cells are shown in the figure. Figure 2 As shown, the image of the stained mature spores of Myospora swimming crab infecting rabbit kidney cells for 1 day, the cytoplasm of free Myospora swimming crab can be observed to proliferate in the cells, and the imaging detection effect is excellent.
[0058] The above results indicate that DiD carbocyanine dyes can trace the proliferation and development process of Mycocystis cynomolgus in rabbit kidney cells.
[0059] From the above experimental results, it can be seen that DiD carbocyanine dyes can be used to track the infection of microsporidia and the proliferation and development process in host cells in real time.
[0060] Comparative Example 1
[0061] A method for fluorescent staining and labeling microsporidia comprises the following steps:
[0062] The intracellular microsporidia in the developmental stage were stained with a staining solution and then observed using a fluorescence microscope.
[0063] The dyeing solution is a solution containing Direct Yellow 96, and its preparation method is as follows:
[0064] 11 mg of 90% pure direct yellow 96 powder was dissolved in 2 mL of 0.1% wt SDS solution, and the mixture was wrapped in tin foil and stored away from light to obtain a direct yellow 96 staining solution mother solution. Before staining, the direct yellow 96 staining solution mother solution was diluted 500 times to prepare a staining solution, thereby obtaining a solution containing direct yellow 96. The concentration of direct yellow 96 in the solution containing direct yellow 96 was 10 μg / mL.
[0065] The microsporidia are Myosporida of Portunus.
[0066] The specific steps of staining microsporidia during the intracellular development period are as follows: cells infected with microsporidia are spread on a cell slide, fixed with a fixative, and then stained with a staining solution and observed using a fluorescence microscope.
[0067] The fixative was 4% w / v paraformaldehyde.
[0068] The steps of staining with staining solution and observation under fluorescence microscope are as follows: collect insect ovarian cells infected with Bombyx mori microspores, centrifuge at 1500g for 5 minutes, wash the surface culture medium with PBS solution and centrifuge again. Resuspend the cells with direct yellow 96 staining solution, maintain at room temperature for 1 minute, centrifuge at 1500g for 5 minutes, wash the cell surface staining solution with PBS solution, repeat twice and resuspend with pure water, add a drop of cell suspension to the slide, blow dry with a hair dryer after spreading, and observe under a wide-field fluorescence microscope.
[0069] Comparative Example 2
[0070] A method for fluorescent staining and labeling microsporidia comprises the following steps:
[0071] The microsporidia spores or microsporidia in the intracellular developmental stage are stained with a staining solution and then observed using a fluorescence microscope.
[0072] Wherein, the dyeing solution is a solution containing direct red 28, and its preparation method is as follows:
[0073] 20 mg of direct red 28 powder was dissolved in 2 mL of buffer solution, and the solution was wrapped in tin foil and stored in dark place to obtain a mother solution of direct red 28 dye solution. Before dyeing, the mother solution of direct red 28 dye solution was diluted 100 times to prepare a dye solution, and a solution containing direct red 28 was obtained. The concentration of direct red 28 in the solution containing direct red 28 was 100 μg / mL.
[0074] The specific steps of staining microsporidia during the intracellular development period are as follows: cells infected with microsporidia are spread on a cell slide, fixed with a fixative, and then stained with a staining solution and observed using a fluorescence microscope.
[0075] The fixative was 4% w / v paraformaldehyde.
[0076] The steps of staining with staining solution and observation under fluorescence microscope are as follows: collect insect ovarian cells infected with Bombyx mori microsporus, resuspend the cells with direct red 28 staining solution, maintain at room temperature for 8 minutes, centrifuge at 1500g for 5 minutes, wash the cell surface staining solution with PBS solution, repeat twice and resuspend with pure water, add a drop of cell suspension to the slide, blow dry with a hair dryer after spreading, observe and photograph under a microscope.
[0077] Comparative Example 3
[0078] A two-color fluorescent labeling method for identifying microsporidia in the schizont multiplication stage and the spore multiplication stage comprises the following steps:
[0079] (1) The blocked cells were used to label the microsporidia in the fission proliferation stage using indirect immunofluorescence using β-Tubulin antibody.
[0080] (2) The cells are then stained with chitin using a fluorescent whitening agent to mark the microsporidia in the spore proliferation stage.
[0081] (3) Observe the samples using a fluorescence microscope.
[0082] The blocked cells are prepared by the following method: firstly, the host cells infected with microsporidia are fixed on a glass slide; then, the fixed cells are immersed in a Triton X-100 solution for permeabilization for 20 minutes; and then, the permeabilized cells are blocked with a cell blocking solution.
[0083] The blocking solution in the above method is a PBS solution containing 0.05% V / V Tween 20, 2% V / V Triton X-100, 0.05% V / V BSA and 10% V / V goat serum.
[0084] The method of using β-Tubulin antibody to label microsporidia in the fission proliferation stage by indirect immunofluorescence is as follows: after washing the closed cells with a PBS solution with a volume fraction of 0.05% Tween 20, the cells are immersed in a primary antibody working solution and incubated at 30°C for 1 hour; the primary antibody working solution is removed, the cells are washed with a PBS solution with a volume fraction of 0.05% Tween 20, and then the cells are immersed in a secondary antibody working solution and incubated at 30°C for 1 hour; the secondary antibody working solution is removed, and then the cells are washed with a PBS solution with a volume fraction of 0.05% Tween 20.
[0085] The primary antibody working solution was a mixture of blocking solution and β-Tubulin antibody diluted at a volume ratio of 1:200. The secondary antibody working solution was a mixture of blocking solution and Alexa Fluor 594-labeled goat anti-mouse IgG diluted at a volume ratio of 1:2000.
[0086] The method of staining chitin with a fluorescent whitening agent to mark microsporidia in the spore proliferation stage is as follows: add 0.1 μg / mL Calcofluor White M2R staining solution to cover the cells and incubate at 30°C for 10 minutes; remove the staining solution, wash with a PBS solution with a volume fraction of 0.05% Tween 20, and then add an anti-fluorescence quenching mounting medium.
[0087] In the steps of Comparative Examples 1 to 3, a fixative is used to fix the cell sample, and the microsporidia are stained on the basis of fixation, which may destroy the structure of the microsporidia and cannot dynamically trace the proliferation and development of the microsporidia. However, the fluorescent labeling method for real-time tracing of the proliferation and development of intracellular microsporidia provided by the present invention does not need to be performed on the basis of cell fixation and permeabilization, does not destroy the cell structure and antigenic epitopes, can accurately reflect the real situation in living cells, and can trace the proliferation and development of microsporidia in real time.
[0088] In Comparative Example 3, two methods or dyes, immunology and fluorescent staining, were used to mark microsporidia at different proliferation stages. However, the fluorescent labeling method for real-time tracing of intracellular microsporidia proliferation and development provided by the present invention only needs to use one dye to label microsporidia at various developmental stages, replacing the traditional complex method based on dual-color or multi-color fluorescent labeling, with simple steps and easy to be combined with other dyes, and has great application potential.
[0089] Therefore, the present invention solves the problem that the existing intracellular microsporidia labeling method cannot simultaneously label various proliferation stages and microsporidia living cells, and cannot perform real-time tracing of their proliferation and development, and fills the gap in the method for continuous tracing of microsporidia intracellular proliferation and development based on living cells.
[0090] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.
[0091] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they understand the basic inventive concepts, and all such changes and modifications fall within the scope of the present invention.
Claims
1. A fluorescent labeling method for real-time tracing of intracellular microsporidia proliferation and development, characterized in that: The steps include: Collecting mature spores of microsporidia; Using a dyeing solution prepared with a lipophilic carbocyanine dye to dye the mature spores of the microsporidia to obtain the dyed mature spores of the microsporidia; The stained mature spores of microsporidia are inoculated into the host cells being cultured for infection to obtain living cell samples; Observing the living cell sample using a laser confocal microscope 5 to 120 hours after infection; The final concentration of the dye solution prepared with the lipophilic carbocyanine dye is 5 μM; The dyeing time is 10 min; The lipophilic carbocyanine dye includes any one of DiI and DiD; The method for obtaining the stained mature spores of microsporidia comprises the following steps: After the mature spores of the microsporidia are centrifuged, the spore precipitate is collected, and the spore precipitate is resuspended by adding the dyeing solution prepared by the lipophilic carbocyanine dye, and then kept at room temperature for 10 minutes; the spore precipitate is centrifuged again, and the spore precipitate is collected and resuspended and washed with a PBS solution to obtain the stained mature spores of the microsporidia.
2. The fluorescent labeling method for real-time tracing of intracellular microsporidia proliferation and development according to claim 1, characterized in that: The centrifugation condition was 8000 g for 5 min.
3. The fluorescent labeling method for real-time tracing of intracellular microsporidia proliferation and development according to claim 1, characterized in that: The concentration of PBS solution was 0.01 M and the pH was 7.
3.
4. The fluorescent labeling method for real-time tracing of intracellular microsporidia proliferation and development according to claim 1, characterized in that: The method of inoculating the stained mature spores of microsporidia into the host cells being cultured comprises the following steps: When the host cells being cultured reach a confluence of 80%, inoculating the stained mature spores of the microsporidia into the host cells being cultured for co-culturing; The co-culture conditions were 37° C., 5% CO 2 , and 95% humidity.
5. The fluorescent labeling method for real-time tracing of intracellular microsporidia proliferation and development according to claim 4, characterized in that: The host cells are rabbit kidney cells.
6. The fluorescent labeling method for real-time tracing of intracellular microsporidia proliferation and development according to claim 1, characterized in that: The microsporidia are Myosporida swimming crab.