A method of observing the surface structure of the nucleus of a cell
By combining pretreatment with nocodazole and cytochalasin B with gentle centrifugation, the problem of structural damage during nucleocytoplasmic separation was solved, realizing an efficient and simplified method for separating cell nuclei while maintaining the integrity of the nucleus for easy observation.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- PEKING UNIV
- Filing Date
- 2025-12-31
- Publication Date
- 2026-05-29
Smart Images
Figure CN122104592A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of cell imaging technology, and in particular to a method for observing the structure of the cell nucleus surface. Background Technology
[0002] In cell biology research, observing the ultrastructure of the cell nucleus, such as using a scanning electron microscope (SEM), is an important means of analyzing its function and state. Before performing such observations, it is usually necessary to isolate and purify the cell nucleus from the intact cell.
[0003] A common method in existing technologies is nucleocytoplasmic separation. This method typically involves treating cells with a hypotonic solution to induce swelling and lysis, then using mechanical methods such as homogenizers to break the cells and release the nuclei, and finally purifying them using methods such as density gradient centrifugation.
[0004] However, this method has significant technical drawbacks. First, the mechanical shearing forces and drastic changes in osmotic pressure caused by the hypotonic environment during homogenization easily damage the original three-dimensional structure of the cell nucleus, leading to shrinkage and deformation of its surface. This morphological damage severely affects the accuracy and realism of subsequent scanning electron microscopy observations, making it impossible to accurately resolve the ultrastructure of the cell nucleus surface. Furthermore, this traditional method is cumbersome, time-consuming, and has low overall operational efficiency.
[0005] Therefore, how to develop a separation method that can effectively maintain the integrity of the three-dimensional structure of the cell nucleus while simplifying the operation process is a technical problem that urgently needs to be solved in this field. Summary of the Invention
[0006] To address the problems existing in the prior art, the present invention provides a method for observing the structure of the cell nucleus surface.
[0007] In a first aspect, the present invention provides a method for separating the nucleus and cytoplasm of a cell, comprising: cell pretreatment, and separating the cytoplasm and the nucleus; The cell pretreatment includes: culturing the cells in a culture medium containing nocodazole and cytochalasin B for 1.5 to 3 hours; the culture medium comprises, by weight, 0.1 to 0.5 parts of nocodazole and 2 to 10 parts of cytochalasin B.
[0008] Traditional nucleocytoplasmic separation methods primarily rely on centrifugation. However, due to the fragility of the cell nucleus, gentler methods such as sucrose gradient centrifugation or citrate buffering are often used, or differential centrifugation is employed to minimize nuclear damage during separation. This invention, based on extensive research, provides a method for pretreating cells with nocodazole and cytochalasin B. After treatment, the binding between the cell nucleus and the cell itself is weakened, while the nucleus remains largely unaffected. This method improves nucleocytoplasmic separation efficiency while preserving the integrity of the nuclear structure.
[0009] Furthermore, the culture medium comprises: nocodazole 100~500 ng / mL and cytochalasin B 2~10 μg / mL; Preferably, the culture medium comprises: nocodazole 200~300 ng / mL and cytochalasin B 4~8 μg / mL.
[0010] Furthermore, the culture conditions for the cell pretreatment include: culture at 35-40°C and 4-8% CO2 concentration; Preferably, the culture is carried out at 36-38°C and 4%-6% CO2 concentration.
[0011] Furthermore, the cells are somatic cells; Preferably, the cells are tumor cells or fibroblasts; More preferably, the cells are HeLa cells or HDF cells.
[0012] Furthermore, when the cells are HeLa cells, the culture medium is DMEM containing 8-20% fetal bovine serum (FBS).
[0013] Furthermore, the separation of cytoplasm and nucleus is performed by centrifugation, and the centrifugation conditions include centrifugation at 0~8℃ and 1000~2000g for 10~20 minutes.
[0014] Furthermore, the centrifugation process includes: placing a substrate in a centrifuge tube, and placing a cell nucleus collection device on the substrate; A cell carrier device is placed directly above the cell nucleus collection device; culture medium is added to both the cell nucleus collection device and the cell carrier device, and after centrifugation, the cell nuclei of the cells in the cell carrier device are detached and collected by the cell nucleus collection device.
[0015] Furthermore, the cell nucleus collection device may be a glass slide, such as a glass slide soaked in polylysine or an organosilicon slide.
[0016] Furthermore, the cell-carrying device is a glass slide.
[0017] Secondly, the present invention provides a method for observing the surface structure of a cell nucleus, comprising: The aforementioned method was used for nucleo-cytoplasmic separation; Observe the cell nucleus; Preferably, the cell nucleus is observed using a light microscope and / or an electron microscope.
[0018] Thirdly, the present invention provides the application of the aforementioned nucleocytoplasmic separation method in improving the structural integrity of the cell nucleus after nucleocytoplasmic separation.
[0019] Fourthly, the present invention provides the use of a combination of nocodazole and cytochalasin B in the preparation of a kit for improving the structural integrity of the nucleus after nucleocytoplasmic separation.
[0020] The present invention has the following beneficial effects: This invention provides a method for nucleocytoplasmic separation. Pretreatment of cells with nocodazole and cytochalasin B effectively improves separation efficiency. Furthermore, this process causes minimal damage to the sample; the cell nuclei collected after pretreatment and centrifugation retain their original structure, mitigating the problems of nuclear shrinkage and collapse, and maximizing the preservation of nuclear structural integrity. This facilitates the observation of the ultrastructure on the cell nucleus surface and has high application value. Attached Figure Description
[0021] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.
[0022] Figure 1 This is a centrifugation diagram provided in Embodiment 1 of the present invention.
[0023] Figure 2 This is a diagram showing the results of light microscopic observation of cell nuclei provided in Embodiment 1 of the present invention.
[0024] Figure 3 This is a scanning electron microscope image of the separated HeLa cell nuclei provided in Embodiment 1 of the present invention.
[0025] Figure 4 This is a scanning electron microscope image of the isolated HDF cell nuclei provided in Embodiment 1 of the present invention.
[0026] Figure 5 This is a scanning electron microscope image of the HeLa cell surface provided in Comparative Example 1 of this invention.
[0027] Figure 6This is a diagram showing the results of observing the separated HeLa cell nuclei using scanning electron microscopy, based on the conventional cell nucleus separation method (hypotonic lysis and cell homogenization, followed by sucrose density centrifugation) provided in Comparative Example 2 of this invention. Detailed Implementation
[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.
[0029] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.
[0030] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available.
[0031] The DMEM culture medium used in the following examples is commercially available, for example, from Gibco.
[0032] Example 1 In this embodiment, a method for observing the microstructure of the cell nuclear surface includes: 1. Nucleocytoplasmic separation (1) First, culture somatic cells, such as HeLa cells (human cervical cancer cell line) or HDF cells (human skin fibroblasts), on a glass slide of appropriate size (using DMEM medium supplemented with 10% fetal bovine serum FBS). The width of the glass slide should be sufficient to fit into the centrifuge tubes in the following steps. If the glass slide is too wide, it needs to be cut to a suitable size for cell culture. Before use, the glass slide needs to be washed with strong acid and soaked in 75% alcohol. Before cell passage, the glass slide needs to be removed, dried with alcohol, and sterilized under ultraviolet light before cell passage. After the somatic cells have adhered to the wall at a suitable density, proceed with the subsequent operations.
[0033] (2) Add Nocodazole to the cells at a final concentration of 250 ng / ml and cytochalasin B at 5 μg / ml, and incubate at 37°C in a 5% CO2 incubator for 2 hours. Follow the instructions... Figure 1Prepare horizontal centrifuge tubes; standard 15 ml centrifuge tubes can be used. Place an acrylic column at the bottom of the centrifuge tube for support. Place a clean glass slide for receiving cell nuclei onto the flat surface of the acrylic column. This slide can be a round glass slide soaked in poly-L-lysine or a silicone slide. The recommended size for silicone slides is 9 mm × 9 mm. After placing the slides, add cell culture medium (DMEM solution with 10% fetal bovine serum).
[0034] (3) Take out the prepared cell-coated slide and place it into a centrifuge tube at an angle, ensuring that the cell-coated side faces downwards. This way, during centrifugation, the cell nuclei can be flung onto the slide below. Make sure the cell-coated slide is submerged in the culture medium to prevent the cells from dehydrating in the air.
[0035] (4) Place the centrifuge tube in a horizontal centrifuge, centrifuge at 4°C and 1500 g for 15 min. The cell nuclei will be attached to the glass slide below. The glass slide containing the cell nuclei can be used for light microscopy (the glass slide for collecting cell nuclei must be a transparent glass slide) or scanning electron microscopy (the glass slide for collecting cell nuclei can be a glass slide or an organosilicon slide).
[0036] 2. Observation of cell nuclei (1) Using a light microscope, slides containing cells and slides from which cell nuclei were collected after centrifugation were fixed with 4% paraformaldehyde. The fixative was then washed off with PBS. The slides were then fixed with mounting medium containing DAPI. After the mounting medium dried, microscopic observation was performed. Images of DAPI-stained cell nuclei under UV light and cells under bright field were collected. The cell nuclei isolated from the cells can be seen in the light microscope images. Figure 2 (As indicated by the white arrow).
[0037] (2) To observe cell nucleus samples using a scanning electron microscope, first remove the glass slide or organosilicon slide containing the cell nuclei, fix it in 2.5% glutaraldehyde solution at room temperature for 1 hour or at 4°C overnight, then wash it three times with 0.1 M dimethyl arsenate, fix it with 1% osmium tetroxide for 30 minutes, wash it three times with deionized water, stain it with 1% uranium acetate for 30 minutes, wash it three times with deionized water, then dehydrate it with a gradient of 30%, 50%, 70%, 90%, and 100% ethanol for 10 minutes each time, and finally treat it with anhydrous ethanol treated with anhydrous copper sulfate for 10 minutes, and then dry it using a carbon dioxide critical point desiccator (LeicaEM CPD300). After drying, spray a 5 nm thick layer of gold using an ion sputtering instrument (Hitachi E-1045), and observe the image under a scanning electron microscope (Hitachi S4800).
[0038] From scanning electron microscopy results ( Figure 3 and Figure 4 As can be seen, the method provided by this invention differs from the traditional method of low-osmotic lysis followed by sucrose centrifugation. Figure 5 Compared to other methods, it does not require high-speed or ultra-high-speed centrifugation (eliminating dependence on high-speed centrifuges), and can separate the cell nucleus of somatic cells relatively quickly. The cell nucleus structure is well preserved, with very little cell nucleus collapse or shrinkage, and the nuclear pore complex structure on the surface of the cell nucleus is well preserved.
[0039] Comparative Example 1 Since the cell nucleus is enclosed by the cell membrane and fixed by the cytoskeleton, if the cells are not pretreated and centrifuged directly, no cell nucleus will be separated from the cell. Even if the centrifugal force is increased, the cells will only be centrifuged off the adherent slide, and the cell nucleus will not be released from the cell separately.
[0040] This invention involves preparing HeLa cells, which are normally cultured on a glass slide, using scanning electron microscopy (without centrifuging the slide), to obtain the following results: Figure 5 The results shown are scanning electron micrographs of HeLa cells cultured on a glass slide without any drug pretreatment or centrifugation. Microvilli on the cell surface are visible, but no isolated nuclei were observed. Even when the slide was centrifuged at 1500 g under the same conditions as in Example 1 (4°C), the nuclei could not be isolated from the cells.
[0041] Comparative Example 2 In the experiment, this invention also selected the method of separating cell nuclei using the traditional method of hypotonic lysis, followed by homogenization using a glass homogenizer, and then preparing samples using scanning electron microscopy.
[0042] The reference method is from the literature: Ori A, Cell type-specific nuclear pores a case in point for context-dependent stoichiometry of molecular machines, Mol Syst Biol. 2013;9:648. The specific supplementary information from this document is on page 11.
[0043] Brief Description: The method employed involves first scraping cultured cells, centrifuging them, lysing them in a hypotonic solution for a period of time, then homogenizing them multiple times using a Dounce homogenizer. The cell nuclei are then collected and centrifuged, washed with a solution, and different concentrations of sucrose solutions are prepared in advance. The high-concentration sucrose solution is placed at the bottom, and the homogenized cell mixture is resuspended using a low-concentration sucrose solution. The mixture is then centrifuged at high speed, the resulting fractions are washed, centrifuged again, and finally resuspended with a solution to obtain the separated cell nuclei. The obtained cell nuclei are then used for scanning electron microscopy.
[0044] Following this method, the cell nucleus was isolated and observed using scanning electron microscopy. The obtained electron microscopic results of the cell nucleus are as follows: Figure 6 As can be seen, the overall morphology of the cell nuclei obtained after using the traditional hypotonic lysis, homogenization, and sucrose density centrifugation is not a full round or elliptical shape. The surface of the cell nuclei has shrunk to a certain extent, and the preservation effect of the ultrastructure of the nuclear pore complex on the surface of the cell nuclei is significantly worse than that in Example 1.
[0045] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims
1. A method for separating the nucleus and cytoplasm of cells, characterized in that, include: Cell pretreatment, and separation of cytoplasm and nucleus; The cell pretreatment includes: culturing the cells in a culture medium containing nocodazole and cytochalasin B for 1.5 to 3 hours; the culture medium comprises, by weight, 0.1 to 0.5 parts of nocodazole and 2 to 10 parts of cytochalasin B.
2. The nucleocytoplasmic separation method according to claim 1, characterized in that, The culture medium comprises: nocodazole 100~500 ng / mL and cytochalasin B 2~10 μg / mL.
3. The nucleocytoplasmic separation method according to any one of claims 1-2, characterized in that, The cell pretreatment culture conditions include: culture at 35-40℃ and 4-8% CO2 concentration.
4. The nucleocytoplasmic separation method according to any one of claims 1-2, characterized in that, The cells in question are somatic cells.
5. The nucleo-cytoplasmic separation method according to claim 4, characterized in that, The culture medium is DMEM containing 8-20% fetal bovine serum.
6. The nucleocytoplasmic separation method according to any one of claims 1-2, characterized in that, The separation of cytoplasm and nucleus is performed by centrifugation, and the centrifugation conditions include centrifugation at 0~8℃ and 1000~2000g for 10~20min.
7. The nucleo-cytoplasmic separation method according to claim 6, characterized in that, The centrifugation process includes: placing a substrate in a centrifuge tube and placing a cell nucleus collection device on the substrate; A cell carrier device is placed directly above the cell nucleus collection device; culture medium is added to both the cell nucleus collection device and the cell carrier device, and after centrifugation, the cell nuclei of the cells in the cell carrier device are detached and collected by the cell nucleus collection device.
8. A method for observing the surface structure of a cell nucleus, characterized in that, include: Nucleus-plasma separation is performed using the method described in any one of claims 1-7; Observe the cell nucleus.
9. The application of the nucleocytoplasmic separation method according to any one of claims 1-7 in improving the structural integrity of the cell nucleus after nucleocytoplasmic separation.
10. Application of the combination of nocodazole and cytochalasin B in the preparation of a kit for improving the structural integrity of the nucleus after nucleocytoplasmic separation.