A fluorescence staining device and a staining method for flow cytometry

Abstract

The application relates to the field of cell staining, in particular to a fluorescence staining device and a staining method for flow cytometry analysis, which comprises a workbench, a feeding cylinder, a mixing cylinder, a nitrogen filling machine, a quantitative feeding component and a cell mixing component. The application can solve the following problems existing in the process of cell staining and marking in the prior art: when viscous cells are stained, the relatively viscous liquid cells are usually difficult to disperse, so that the fluorescence staining liquid can only stain the surface layer of the cell liquid, cannot penetrate into the cell inside located in the surface layer, so that the cells inside the surface layer cannot be stained, and further affect the uniformity of cell staining; when the cells are stained, bacteria or dust and other impurities in the air are easily attached to the surface of the cells, further affecting the detection effect and detection precision, and easily leading to inactivation of the cells after contacting with the air, further affecting the survival rate of the cells.

Description

Technical Field

[0001] This invention relates to the field of cell staining, and in particular to a fluorescent staining apparatus and staining method for flow cytometry analysis. Background Technology

[0002] In biological experiments, in order to study the physiological functions of cells, the occurrence and development of diseases, etc., it is necessary to analyze and detect cells using flow cytometry. By using a variety of high technologies, multiple characteristics and functions of a large number of cells can be analyzed simultaneously in a very short time. Flow cytometry analysis has extremely wide applications in cell biology, hematology, immunology, oncology and other fields.

[0003] Since flow cytometers typically use lasers as the light source, cells need to be stained with fluorescent staining solution before analysis. When irradiated by the laser beam, the fluorescently stained cells produce scattered light and fluorescence, which facilitates the analysis of various parameters of the cells by flow cytometer.

[0004] Cell staining refers to staining cells with antibodies labeled with fluorescent dyes, such as flow cytometry antibodies. This involves injecting the antibody into a flow cytometry tube containing cells, and then mixing the cells evenly with the fluorescent staining solution.

[0005] In the prior art, a large number of cell staining devices and related apparatuses have been disclosed. For example, Chinese Patent No. CN113702142A discloses a cell staining instrument. When in use, reagent tubes are first placed in batches on a reagent rack, and the reagent rack is placed in a reagent compartment holder. Then, the injection needle draws up the reagent and drops it onto the slide rocking assembly. The slide rocking assembly completes the mixing between the cells and the fluorescent staining solution by the oscillation of the slide stage, thereby achieving cell staining.

[0006] In the cell staining process described above, the existing technology uses a swinging mechanism to allow the liquid to flow repeatedly in the cell area of ​​the slide, thereby achieving a mixing effect. At the same time, the liquid will not flow away quickly due to the poor water absorption of the cells. Different tilt angles are used in the antibody addition and washing steps to change the fluidity of the liquid, so that the cells are continuously covered by the liquid, and the reaction time is more sufficient when the slide is kept horizontal.

[0007] However, the following shortcomings exist in the process of staining and labeling cells using the aforementioned existing techniques: 1. Since flow cytometry includes both highly mobile liquids and relatively viscous liquids, the existing techniques described above are only applicable to staining highly mobile liquid cells. When staining relatively viscous liquid cells, the cells are usually clump-like and cannot be dispersed by the oscillation of the slide assembly. Therefore, the fluorescent staining solution can only stain the surface layer of the cell fluid and cannot penetrate into the interior of the relatively viscous liquid cells. Consequently, the antibodies inside the relatively viscous liquid cells are not stained, affecting the uniformity of cell staining.

[0008] 2. Furthermore, since air contains impurities such as bacteria or dust, cell staining must be performed under sterile conditions. However, the existing technology described above cannot ensure that cells are sterile when staining them, making it easy for bacteria or dust from the air to adhere to the cell surface, thus affecting the detection effect and accuracy.

[0009] 3. In addition, stained cells need to be transferred to a cytometer for analysis promptly. However, the existing technology described above can only retain stained cells on the slide rocking assembly, making it difficult to remove cells from the assembly and impossible to quantify cells according to analytical needs. This affects the efficiency of cell detection, and the cells are easily exposed to air, thus affecting their survival rate. Therefore, based on the points stated above, there is room for improvement in existing cell staining techniques. Summary of the Invention

[0010] To address the aforementioned problems, this invention provides a fluorescence staining apparatus for flow cytometry analysis, comprising a worktable with two feed cylinders, a mixing cylinder mounted at the lower end of the worktable, a nitrogen filling machine at the upper end of the worktable with its output end extending into the mixing cylinder, a quantitative feeding component for use with the feed cylinders at the upper end of the worktable, the quantitative feeding component including two extrusion plates located on both sides of the feed cylinders, and a cell mixing component disposed inside the mixing cylinder.

[0011] The cell mixing component includes a storage tank rotatably connected to the lower end of the mixing cylinder. A mixing assembly is provided on the inner wall of the mixing cylinder. The mixing assembly includes an annular toothed ring rotatably connected to the lower side of the inner wall of the mixing cylinder. A driving assembly is installed between the mixing cylinder and the storage tank. The driving assembly includes an idler wheel disposed inside the mixing cylinder and meshing with the annular toothed ring. A discharge assembly is provided on the outer wall of the storage tank near its lower side. The discharge assembly includes a telescopic corrugated pipe.

[0012] Preferably, the quantitative feeding component further includes vertical plates. Two vertical plates are symmetrically arranged at the upper end of the worktable along its length direction. Two threaded rods are symmetrically rotatably connected between the vertical plates along the width direction of the worktable. The two threaded rods are connected by a belt drive. A forward and reverse motor connected to the threaded rod is provided on any one of the vertical plates through a motor cover. Two threads with opposite directions are symmetrically opened on the outer wall of the threaded rod along the feed cylinder. An adjustment and limiting component is provided between the threaded rod and the extrusion plate.

[0013] Preferably, the adjusting and limiting component includes a rectangular sleeve. Multiple rectangular sleeves are fitted onto the outer wall of the threaded rod by means of threaded connection. The rectangular sleeves slide through the extrusion plate. Multiple limiting holes are equally spaced along the axis of the threaded rod on the side of the rectangular sleeves near the middle of the extrusion plate. A receiving groove is formed inside the extrusion plate. A pressure rod is slidably connected inside the receiving groove. A fixed magnetic block is installed on the bottom wall of the receiving groove. A displacement magnetic block is provided at the lower end of the pressure rod to cooperate with the fixed magnetic block.

[0014] Preferably, triangular blocks are installed on both sides of the pressure rod relative to the rectangular sleeve. The inclined surface of the triangular blocks gradually slopes from top to bottom towards the side closer to the middle of the pressure rod. Two circular holes are symmetrically opened inside the extrusion plate along the receiving groove. An actuating contact rod is slidably connected in the circular holes. One end of the actuating contact rod slides and abuts against the inclined surface of the triangular blocks, and the other end is installed with a clamping pin through the connecting plate. The end of the clamping pin away from the connecting plate passes through the extrusion plate and cooperates with the limiting hole. A retraction spring sleeved on the outside of the actuating contact rod is installed between the side of the connecting plate away from the clamping pin and the inner wall of the circular hole.

[0015] Preferably, the mixing component further includes a support rod, the inner wall of the annular toothed ring is provided with a support rod, the lower end of the support rod is equipped with a rotating plate by two support rods symmetrically arranged along its length, the outer wall of the support rod is sleeved with a scraper that slides in contact with the inner side wall of the storage barrel, a smoothing plate is installed in the middle of the lower end of the rotating plate, and two cutting groups are symmetrically arranged along the smoothing plate at the lower end of the rotating plate. The lower end of the flat plate makes rotatable contact with the bottom wall of the storage tank. Both sides of the flat plate in the width direction are chamfered, and the side of the chamfer away from the middle of the flat plate gradually slopes upward.

[0016] Preferably, each cutting group includes multiple actuating blocks arranged at equal intervals along the length of the rotating plate. The lower end of the actuating block slides in contact with the bottom wall of the storage tank. The actuating block is a triangular pyramid structure used to cut the cell fluid and fluorescent staining solution. The multiple actuating blocks in the same cutting group are divided into two groups with the middle of the rotating plate as the center. The two groups of actuating blocks are arranged in a centrally symmetrical manner along the middle of the rotating plate, and the actuating blocks of the two cutting groups are arranged alternately.

[0017] Preferably, the drive assembly further includes a positioning motor. The positioning motor is mounted on the outer wall of the mixing drum via a motor mount. The output shaft of the positioning motor is provided with a first gear and a second gear from top to bottom. The first gear meshes with an idler gear. The outer wall of the storage drum is fitted with a positioning gear ring that meshes with the second gear.

[0018] Preferably, the discharge assembly further includes a pressing plate sleeved on the outer wall of the telescopic corrugated pipe on the side away from the storage tank, a top extension spring sleeved on the outside of the telescopic corrugated pipe is installed between the pressing plate and the outer wall of the storage tank, a first one-way valve is installed inside the telescopic corrugated pipe on the side near the storage tank, a second one-way valve is provided inside the telescopic corrugated pipe on the side away from the storage tank, and a partition is provided between the storage tank and the pressing plate.

[0019] Preferably, the partition includes threaded guide rods. Multiple threaded guide rods are evenly arranged in annular patterns along the telescopic corrugated pipe on the outer wall of the storage tank. The threaded guide rods slide through the pressing plate. Each threaded guide rod has a nut fitted on its outer wall by means of threaded connection. Multiple nuts are connected to each other by a belt.

[0020] Preferably, the upper end of the feed cylinder is provided with a sealing ring to prevent oxygen from entering, the upper half of the feed cylinder is symmetrically provided with two supports connected to the worktable along its axis, the lower half of the feed cylinder extends into the mixing cylinder after passing through the worktable, and the middle part of the feed cylinder is a flexible tube that is easy to deform, and an execution plate that abuts against the flexible tube is installed on the side of the extrusion plate near the feed cylinder.

[0021] In addition, the present invention also provides a fluorescent staining method for flow cytometry analysis, comprising the following steps: S1: adding cell solution and fluorescent staining solution: firstly, nitrogen gas is added into the device through a nitrogen filling machine, and then the cell solution and fluorescent staining solution are added into the mixing cylinder in a certain proportion through a quantitative feeding component; S2: Cell Mixing Staining: The cell solution and fluorescent staining solution in S1 are mixed evenly through the cell mixing component; S3: Quantitative removal of stained cells: The stained cells are quantitatively removed according to the needs of detection and analysis through the discharge component.

[0022] In summary, this application includes at least one of the following beneficial technical effects: I. This invention introduces nitrogen into the mixing cylinder, preventing cells from coming into contact with air during the staining process and avoiding the inactivation of cells by oxygen in the air, thereby improving the cell survival rate during the staining process. In addition, it can prevent bacteria and dust and other impurities from adhering to the cell surface, thus ensuring that the cell staining process is in a sterile state.

[0023] II. This invention uses a rotating plate to drive a smoothing plate and a stirring block to move circumferentially around the axis of the storage tank. The stirring block can cut a crack in the cell fluid, allowing the fluorescent staining solution on the surface of the cell fluid to enter the crack. Then, the smoothing plate smooths the crack, squeezing the fluorescent staining solution inside the crack into the cell fluid. By repeating this step, the cell fluid and fluorescent staining solution can be repeatedly cut and smoothed, thereby fully mixing the cell fluid and fluorescent staining solution and improving the overall staining of the cell fluid.

[0024] Third, the present invention can adjust the squeezing force applied by the execution plate to the flexible tube by moving the squeezing plate to the limiting hole at different positions on the rectangular sleeve; since the squeezing forces on the two flexible tubes are different, the flow rates of cell fluid and fluorescent staining solution through the flexible tubes are different; thereby controlling the cell fluid and fluorescent staining solution to be added to the mixing cylinder in a certain proportion, so as to enhance the staining effect of the cells.

[0025] Fourth, this invention allows the cell fluid to be discharged by pushing and releasing the pressing plate, which causes the telescopic corrugated pipe to draw the cell fluid from the storage tank into its interior through the first one-way valve and discharge it into the external flow pipe through the second one-way valve. In addition, the movement distance of the pressing plate can be controlled by adjusting the position of the nut, thereby controlling the squeezing amplitude of the telescopic corrugated pipe and thus controlling the quantitative discharge of cell fluid in the telescopic corrugated pipe. The operation is convenient.

[0026] Fifth, this invention uses a support rod to drive a support rod and a scraper to rotate along the inner wall of the storage tank, which can scrape off the cell fluid and fluorescent staining solution adhering to the inner wall of the storage tank and drop them to the bottom of the storage tank, thereby thoroughly stirring and mixing the cell fluid and fluorescent staining solution in the storage tank, thus ensuring the stirring effect.

[0027] VI. In this invention, the engagement of gear one, idler gear, and ring gear drives the support rod, rotating plate, smoothing plate, and actuating block to rotate clockwise synchronously. At the same time, gear two drives the storage tank to rotate counterclockwise via the positioning ring gear. The storage tank drives the cell sap and fluorescent staining solution at its bottom to rotate counterclockwise, thereby causing the smoothing plate and actuating block to rotate in opposite directions with the storage tank, which enhances the mixing intensity of the cell sap and fluorescent staining solution by the smoothing plate and actuating block. Attached Figure Description

[0028] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0029] Figure 1 This is a first structural schematic diagram of the present invention.

[0030] Figure 2 This is a schematic diagram of the second structure of the present invention.

[0031] Figure 3This is a schematic diagram of the structure between the feed cylinder and the quantitative feed component of the present invention.

[0032] Figure 4 This is a schematic diagram of the structure between the mixing cylinder and the cell mixing component of the present invention.

[0033] Figure 5 This is the present invention. Figure 4 A magnified view of a portion at point F.

[0034] Figure 6 This is a schematic diagram of the structure of the hybrid component and the driving component of the present invention.

[0035] Figure 7 This is a schematic diagram of the structure of the hybrid component of the present invention.

[0036] Figure 8 This is the present invention. Figure 7 A magnified view of a portion of point M.

[0037] Figure 9 This is a schematic diagram of the quantitative feeding component of the present invention.

[0038] Figure 10 This is the present invention. Figure 9 A magnified view of a portion of point G.

[0039] Figure 11 This is a schematic diagram of the structure of the adjusting limit component of the present invention.

[0040] Figure 12 This is a schematic diagram of the material discharge assembly of the present invention. In the diagram, 1 is a workbench; 2 is a feed cylinder; 21 is a sealing ring; 22 is a flexible tube; 3 is a mixing cylinder; 4 is a nitrogen filling machine; 5 is a quantitative feeding component; 51 is an extrusion plate; 511 is an execution plate; 52 is a vertical plate; 53 is a threaded rod; 54 is a forward / reverse motor; 55 is an adjusting limit assembly; 551 is a rectangular sleeve; 552 is a limit hole; 553 is a pressure rod; 554 is a fixed magnetic block; 555 is a displacement magnetic block; 556 is a triangular block; 557 is an execution contact rod; 558 is a clamping pin; 559 is a contraction spring; 6 is a cell mixing component; 61 is a storage tank; and 62 is a mixing assembly. Components; 621, Ring gear; 622, Support rod; 623, Support rod; 624, Scraper; 625, Rotating plate; 626, Smoothing plate; 627, Cutting assembly; 628, Actuating block; 63, Drive assembly; 631, Idler wheel; 632, Positioning motor; 633, Gear No. 1; 634, Gear No. 2; 635, Positioning gear; 64, Discharge assembly; 641, Telescopic bellows; 642, Pressing plate; 643, Top extension spring; 644, First check valve; 645, Second check valve; 646, Partition; 647, Threaded guide rod; 648, Nut. Detailed Implementation

[0041] The following is in conjunction with the appendixFigures 1-12 The embodiments of the present invention will be described in detail, but the present invention may be implemented in many different ways as defined and covered by the claims.

[0042] This application discloses a fluorescent staining device for flow cytometry analysis. The device is primarily used for fluorescent staining of flow cytometry cells. Technically, it ensures that cells do not come into contact with air during staining, preventing air from affecting cell viability and ensuring the cells remain sterile. Specifically, during staining, the device repeatedly cuts and smooths the cell solution and fluorescent staining solution, ensuring thorough mixing and complete staining. Furthermore, the device controls the flow rate of the cell solution and fluorescent staining solution to ensure they are added to the mixing chamber in a specific ratio; and it allows the stained cell solution to be quantitatively discharged according to the requirements of the analysis.

[0043] Example 1: Reference Figure 1 and Figure 2 As shown, a fluorescence staining device for flow cytometry analysis includes a workbench 1 with two feed cylinders 2 on it. A mixing cylinder 3 is installed at the lower end of the workbench 1, and a nitrogen filling machine 4 is installed at the upper end of the workbench 1. The output end of the nitrogen filling machine 4 extends into the mixing cylinder 3. A quantitative feeding component 5 is installed at the upper end of the workbench 1 to work with the feed cylinders 2. The quantitative feeding component 5 includes two extrusion plates 51 located on both sides of the feed cylinders 2. A cell mixing component 6 is installed inside the mixing cylinder 3.

[0044] In practical applications, the nitrogen filling machine 4 is first started, adding nitrogen gas into the mixing cylinder 3. This prevents the cells from coming into contact with air during staining, avoiding the inactivation of cells by oxygen in the air and thus improving the cell survival rate during staining. In addition, it can prevent bacteria and dust from adhering to the cell surface, thus ensuring a sterile environment during cell staining. Then, the cell solution and fluorescent staining solution are added to different feed cylinders 2, and then the quantitative feed component 5 adds the cell solution and fluorescent staining solution to the mixing cylinder 3 in proportion. Subsequently, the cell mixing component 6 mixes the cell solution and fluorescent staining solution in the mixing cylinder 3 evenly. Finally, the stained cell solution is quantitatively removed according to the detection and analysis requirements.

[0045] Reference Figure 2 and Figure 3As shown, since a certain amount of cells need to be stained according to the requirements of the detection and analysis, in order to prevent waste of cell solution, the cell solution needs to be quantitatively added to the mixing cylinder 3. Based on this, the present invention provides a quantitative feeding component 5. Specifically, the quantitative feeding component 5 also includes a vertical plate 52. Two vertical plates 52 are symmetrically arranged along the length direction of the upper end of the worktable 1. Two threaded rods 53 are symmetrically rotatably connected between the vertical plates 52 along the width direction of the worktable 1. The two threaded rods 53 are connected by a belt drive. A forward and reverse motor 54 connected to the threaded rod 53 is provided on any one of the vertical plates 52 through a motor cover. Two threads with opposite directions are symmetrically opened on the outer wall of the threaded rod 53 along the feeding cylinder 2. An adjustment and limiting component 55 is provided between the threaded rod 53 and the extrusion plate 51.

[0046] In this embodiment, the threaded rod 53 is made bidirectional by the threaded rod 53 with the opposite direction of its outer wall. When the threaded rod 53 rotates, the threaded rod 53 can drive the extrusion plates 51 on both sides of the feed cylinder 2 to move relative to each other or move away from each other by adjusting the limiting component 55.

[0047] Continue to refer to Figure 2 and Figure 3 As shown, further, in this embodiment, a sealing ring 21 for preventing oxygen from entering is provided at the upper end of the feed cylinder 2. The sealing ring 21 can prevent external air from entering the feed cylinder 2 at will, but will not affect the addition of cell fluid or fluorescent staining solution into the feed cylinder 2. The upper half of the feed cylinder 2 is symmetrically provided with two supports connected to the workbench 1 along its axis. The lower half of the feed cylinder 2 extends into the mixing cylinder 3 after passing through the workbench 1. The middle part of the feed cylinder 2 is a flexible tube 22 that is easy to deform. The flexible tube 22 can undergo elastic deformation under the action of external force and can automatically recover when there is no external force. An execution plate 511 that abuts against the flexible tube 22 is installed on the side of the extrusion plate 51 near the feed cylinder 2.

[0048] In the specific implementation process, the forward and reverse motor 54 is started. The forward and reverse motor 54 drives the extrusion plates 51 on both sides of the feed cylinder 2 to move relative to each other through the threaded rod 53. During the movement of the extrusion plates 51, the execution plate 511 applies extrusion force to the flexible tube 22, causing the flexible tube 22 to deform. When the deformation of the flexible tube 22 is large, the inner diameter of the flexible tube 22 is relatively reduced, thereby reducing the flow rate of cell fluid or fluorescent staining solution passing through the flexible tube 22, thus reducing the flow rate of cell fluid or fluorescent staining solution.

[0049] Conversely, when the squeezing force applied by the execution plate 511 to the flexible tube 22 is small, the deformation of the flexible tube 22 is relatively small. At this time, the inner diameter of the flexible tube 22 is relatively increased, which can increase the flow rate of the cell fluid or fluorescent staining solution passing through the flexible tube 22, thereby increasing the flow rate of the cell fluid or fluorescent staining solution. Thus, by controlling the flow rate of the cell fluid or fluorescent staining solution, both can be quantitatively added into the mixing cylinder 3.

[0050] Reference Figure 2 and Figure 4 As shown, although the cell sap and fluorescent staining solution can be initially mixed after being placed in the mixing cylinder 3, the uniformity of the mixture cannot be guaranteed. Therefore, in order to ensure that the cells are fully stained, the cell sap and fluorescent staining solution in the mixing cylinder 3 need to be mixed evenly. Specifically, the cell mixing component 6 includes a storage tank 61 rotatably connected to the lower end of the mixing cylinder 3, a mixing assembly 62 is provided on the inner wall of the mixing cylinder 3, the mixing assembly 62 includes an annular toothed ring 621 rotatably connected to the lower side of the inner wall of the mixing cylinder 3, a driving assembly 63 is installed between the mixing cylinder 3 and the storage tank 61, and a discharge assembly 64 is provided on the outer wall of the storage tank 61 near its lower side.

[0051] In the specific implementation process, the driving component 63 drives the annular toothed ring 621 to rotate. The annular toothed ring 621 mixes and stirs the cell fluid and fluorescent staining solution inside the storage tank 61 through the mixing component 62, thereby ensuring the uniformity of cell staining.

[0052] Reference Figure 4 , Figure 6 , Figure 7 and Figure 8 As shown, this example also provides a mixing assembly 62 for stirring and mixing the cell fluid and fluorescent staining solution inside the storage tank 61. Specifically, the mixing assembly 62 also includes a support rod 622. The inner wall of the annular toothed ring 621 is provided with the support rod 622. The lower end of the support rod 622 is equipped with a rotating plate 625 through two support rods 623 symmetrically arranged along its length. The outer wall of the support rod 623 is fitted with a scraper 624 that slides in contact with the inner side wall of the storage tank 61. A smoothing plate 626 is installed in the middle of the lower end of the rotating plate 625, and two cutting groups 627 are symmetrically arranged along the smoothing plate 626 at the lower end of the rotating plate 625.

[0053] The lower end of the flat plate 626 is in rotatable contact with the inner bottom wall of the storage bucket 61. Both sides of the flat plate 626 in the width direction are provided with chamfers, and the side of the chamfer away from the middle of the flat plate 626 gradually slopes upward.

[0054] In the specific implementation process, the annular toothed ring 621 drives the rotating plate 625 to rotate through the supporting rod 622 and the support rod 623. The rotating plate 625 drives the smoothing plate 626 to rotate synchronously. During the rotation of the rotating plate 625 and the smoothing plate 626, the cell fluid and fluorescent staining solution at the bottom of the storage tank 61 can be stirred and mixed. During this period, the support rod 623 drives the scraper 624 to scrape off the cell fluid and fluorescent staining solution attached to the inner wall of the storage tank 61 and drop it to the bottom of the storage tank 61, thereby thoroughly stirring and mixing the cell fluid and fluorescent staining solution in the storage tank 61, thus ensuring the stirring effect.

[0055] Furthermore, in this embodiment, when staining viscous cell fluid, the viscous cell fluid is in a flocculent state, so the fluorescent staining solution can only stain the cells on the surface of the cell fluid, thus the cells inside the cell fluid are not stained, affecting the uniformity of staining; to solve this problem, the present invention provides a toggle block 628, specifically: Each cutting group 627 includes multiple actuating blocks 628 arranged at equal intervals along the length of the rotating plate 625. The lower end of the actuating block 628 slides in contact with the inner bottom wall of the storage tank 61. The actuating block 628 is a triangular pyramid structure used to cut the cell sap and fluorescent staining solution. The multiple actuating blocks 628 in the same cutting group 627 are divided into two groups with the middle of the rotating plate 625 as the center. The two groups of actuating blocks 628 are arranged in a centrally symmetrical manner along the middle of the rotating plate 625. When the rotating plate 625 drives the actuating blocks 628 to rotate synchronously, the V-shaped surface of the actuating block 628 used to cut the cell sap and fluorescent staining solution is in the same direction as its movement, so that the actuating block 628 can always cut the cell sap and fluorescent staining solution at the bottom of the storage tank 61. Moreover, the actuating blocks 628 of the two cutting groups 627 are arranged in an alternating manner, which makes it easier to increase the cutting area of ​​the actuating block 628 on the cell sap and fluorescent staining solution, thereby increasing the contact area between the cell sap and fluorescent staining solution.

[0056] In the specific implementation process, the rotating plate 625 drives the actuating block 628 to rotate synchronously, so that the actuating block 628 moves circumferentially around the axis of the storage tank 61. During this period, the actuating block 628 can cut a crack in the cell fluid, allowing the fluorescent staining solution on the surface of the cell fluid to enter the crack, so that the fluorescent staining solution can stain the cells on the inner wall of the crack. Then the smoothing plate 626 smooths the crack, so that the cell fluid and fluorescent staining solution return to their original state. At this time, the fluorescent staining solution in the crack is squeezed into the cell fluid. By repeating this step, the repeated cutting and smoothing between the cell fluid and the fluorescent staining solution can be achieved, so as to fully mix the cell fluid and the fluorescent staining solution, thereby improving the overall staining of the cell fluid.

[0057] Reference Figure 5 and Figure 6As shown, the drive assembly 63 includes an idler wheel 631 disposed inside the mixing cylinder 3 and meshing with the annular gear ring 621. The drive assembly 63 also includes a positioning motor 632. The positioning motor 632 is disposed on the outer wall of the mixing cylinder 3 through a motor base. The output shaft of the positioning motor 632 is provided with a first gear 633 and a second gear 634 from top to bottom. The first gear 633 meshes with the idler wheel 631. The outer wall of the storage tank 61 is fitted with a positioning gear ring 635 that meshes with the second gear 634.

[0058] In the specific implementation process, the positioning motor 632 is started, which drives the first gear 633 and the second gear 634 to rotate clockwise synchronously. The first gear 633 drives the ring gear 621 to rotate clockwise through the idler wheel 631. The ring gear 621 drives the rotating plate 625, the smoothing plate 626 and the actuating block 628 to rotate clockwise synchronously through the support rod 622. At the same time, the second gear 634 drives the storage tank 61 to rotate counterclockwise through the positioning gear 635. The storage tank 61 drives the cell fluid and fluorescent staining solution at its bottom to rotate counterclockwise. Thus, the smoothing plate 626 and the actuating block 628 rotate in opposite directions with the storage tank 61, so as to enhance the mixing intensity of the cell fluid and fluorescent staining solution by the smoothing plate 626 and the actuating block 628.

[0059] Example 2: Reference Figure 9 , Figure 10 and Figure 11 As shown, based on Example 1, to ensure the staining effect and uniformity of cells, the cell solution and fluorescent staining solution need to be mixed in a certain proportion. Therefore, the cell solution and fluorescent staining solution need to be added to the mixing cylinder 3 in a certain proportion. Furthermore, although the extrusion plate 51 can control the flow rate of the cell solution and fluorescent staining solution inside the flexible tube 22 by extruding the execution plate 511, it cannot control the proportionate addition of the cell solution and fluorescent staining solution to the mixing cylinder 3. Therefore, it is necessary to extrude the flexible tube 22 containing the cell solution and fluorescent staining solution differently to control the different flow rates of the cell solution and fluorescent staining solution, thereby ensuring that the cell solution and fluorescent staining solution are added to the mixing cylinder 3 in a proportionate manner. Based on this, the present invention provides an adjusting limiting component 55, specifically: The adjusting limit assembly 55 includes a rectangular sleeve 551. Multiple rectangular sleeves 551 are fitted onto the outer wall of the threaded rod 53 by means of threaded connection. The rectangular sleeves 551 slide through the extrusion plate 51. During the rotation of the threaded rod 53, the rectangular sleeves 551 can drive the extrusion plate 51 to move relative to or away from each other. Multiple limiting holes 552 are equally spaced along the axis of the threaded rod 53 on one side of the rectangular sleeve 551 near the middle of the extrusion plate 51. A receiving groove is opened inside the extrusion plate 51. A pressure rod 553 is slidably connected inside the receiving groove. A fixed magnetic block 554 is installed on the bottom wall of the receiving groove. A displacement magnetic block 555 is provided at the lower end of the pressure rod 553 to cooperate with the fixed magnetic block 554.

[0060] It should be noted that the fixed magnetic block 554 and the displacement magnetic block 555 attract each other. When the pressure rod 553 is pressed down, the pressure rod 553 drives the displacement magnetic block 555 to attract the fixed magnetic block 554, which facilitates the limiting of the pressure rod 553 and prevents the pressure rod 553 from moving upward at will.

[0061] Furthermore, in this embodiment, triangular blocks 556 are installed on both sides of the pressure rod 553 relative to the rectangular sleeve 551. The inclined surface of the triangular blocks 556 gradually tilts from top to bottom towards the side closer to the middle of the pressure rod 553. Two circular holes are symmetrically opened inside the extrusion plate 51 along the receiving groove. An actuating contact rod 557 is slidably connected in the circular holes. One end of the actuating contact rod 557 slides and abuts against the inclined surface of the triangular blocks 556, and the other end is installed with a clamping pin 558 through the connecting plate. The end of the clamping pin 558 away from the connecting plate passes through the extrusion plate 51 and cooperates with the limiting hole 552. A retraction spring 559 sleeved on the outside of the actuating contact rod 557 is installed between the side of the connecting plate away from the clamping pin 558 and the inner wall of the circular hole.

[0062] In this embodiment, the retraction spring 559 always applies a retraction force to the connecting plate pointing towards the pressure rod 553, causing the connecting plate and the clamping pin 558 to retract into the circular hole in the initial state (in... Figure 11 (As shown in the figure), at this time, there is a movable connection between the extrusion plate 51 and the rectangular sleeve 551.

[0063] In the specific implementation process, the extrusion plate 51 is pushed, causing the extrusion plate 51 to move the execution plate 511 along the length of the rectangular sleeve 551. Then, the pressure rod 553 is pressed, and the pressure rod 553 drives the triangular block 556 to move synchronously. The triangular block 556 applies a pushing force to the execution contact rod 557 on the side away from the pressure rod 553. The execution contact rod 557 drives the clamping pin 558 to be inserted into the limiting hole 552 through the connecting plate. Figure 10 (as shown in the figure), which facilitates the limiting and fixing of the extrusion plate 51. At this time, the extrusion plate 51 and the rectangular sleeve 551 are relatively fixed.

[0064] Since the rectangular sleeve 551 has multiple limiting holes 552 on its side wall, the magnitude of the extrusion force applied by the actuator plate 511 to the flexible tube 22 can be adjusted by moving the extrusion plate 51 to the limiting holes 552 at different positions on the rectangular sleeve 551.

[0065] Subsequently, the extrusion plates 51 on both sides of the feed tube 2 move relative to each other and drive the execution plate 511 to apply extrusion force to the flexible tube 22, thereby controlling the flow rate of cell fluid or fluorescent staining solution through the flexible tube 22. Since the extrusion forces on the two flexible tubes 22 are different, the flow rates of cell fluid and fluorescent staining solution through the flexible tube 22 are different. This controls the cell fluid and fluorescent staining solution to be added into the mixing tube 3 in a certain proportion, so as to enhance the staining effect of the cells.

[0066] Example 3: Reference Figure 12 As shown, based on Embodiment 1, since only a certain amount of cells are needed for detection and analysis, it is necessary to quantitatively extract the stained cell solution from the storage tank 61 according to the requirements of detection and analysis. Therefore, the present invention provides a discharge assembly 64. Specifically, the discharge assembly 64 includes a telescopic corrugated pipe 641 installed on the lower side of the outer wall of the storage tank 61. The discharge assembly 64 also includes a pressing plate 642 sleeved on the outer wall of the telescopic corrugated pipe 641 away from the storage tank 61. A top extension spring 643 sleeved on the outside of the telescopic corrugated pipe 641 is installed between the pressing plate 642 and the outer wall of the storage tank 61. A first one-way valve 644 is installed inside the telescopic corrugated pipe 641 on the side close to the storage tank 61. A second one-way valve 645 is provided inside the telescopic corrugated pipe 641 on the side away from the storage tank 61. A partition 646 is provided between the storage tank 61 and the pressing plate 642.

[0067] In this embodiment, the first one-way valve 644 can only discharge the cell fluid inside the storage tank 61 to the outside, and cannot allow external air or cell fluid to enter the storage tank 61; the second one-way valve 645 can only discharge the cell fluid inside the telescopic bellows 641, and cannot allow external air to enter the telescopic bellows 641; in addition, the top extension spring 643 always applies a pushing force to the pressing plate 642 away from the storage tank 61, so that the distance between the pressing plate 642 and the storage tank 61 is maximized in the initial state.

[0068] In the specific implementation process, the pressing plate 642 is pushed, causing it to move closer to the storage tank 61. At this time, the air inside the telescopic bellows 641 is discharged through the second one-way valve 645. Then, the pressing plate 642 is released, and under the action of the extension spring 643, it returns to its initial state, creating a negative pressure inside the telescopic bellows 641. The stained cell fluid inside the storage tank 61 is then drawn out into the telescopic bellows 641 through the first one-way valve 644. The pressing plate 642 is then pushed again, and a flow meter is placed below the end of the telescopic bellows 641 away from the storage tank 61. The pressing plate 642 squeezes the telescopic bellows 641 and forces the cell fluid inside through the second one-way valve 645 into the flow meter, thus completing the discharge of the cell fluid.

[0069] Furthermore, since the force cannot be controlled when pushing the pressing plate 642, the extruded cell fluid is easily too much or too little when squeezing the telescopic corrugated tube 641, making it impossible to accurately control the output of cell fluid. Based on this, in this embodiment, the partition 646 includes a threaded guide rod 647. Multiple annularly distributed threaded guide rods 647 are evenly arranged along the telescopic corrugated tube 641 on the outer wall of the storage tank 61. The threaded guide rods 647 slide through the pressing plate 642. Each threaded guide rod 647 has a nut 648 sleeved on its outer wall through a threaded connection. Multiple nuts 648 are connected by a belt. Rotating any one nut 648 can drive the other nuts 648 to rotate synchronously.

[0070] In the specific implementation process, the nut 648 is rotated and moves along the axis of the threaded guide rod 647 to adjust the position of the nut 648; when the pressing plate 642 is pushed, the pressing plate 642 abuts against the nut 648, thereby controlling the moving distance of the pressing plate 642, and thus controlling the squeezing amplitude of the telescopic bellows 641, thereby controlling the quantitative discharge of cell fluid in the telescopic bellows 641, which is convenient to operate.

[0071] Furthermore, if less cell fluid needs to be extracted, the moving distance of the pressing plate 642 will decrease accordingly, and the squeezing amplitude of the telescopic bellows 641 will also decrease accordingly. At this time, less cell fluid is drawn out of the storage tank 61 by the telescopic bellows 641, so the telescopic bellows 641 needs to be squeezed repeatedly until the telescopic bellows 641 is filled with enough squeezed cell fluid.

[0072] In addition, the present invention also provides a fluorescence staining method for flow cytometry analysis, comprising the following steps: S1: Add cell solution and fluorescent staining solution: First, start the nitrogen filling machine 4. The nitrogen filling machine 4 adds nitrogen gas into the mixing cylinder 3 so that the cells do not come into contact with air during the staining process, avoiding the inactivation of cells by oxygen in the air, thereby improving the cell survival rate during the staining process; in addition, it can ensure that the cell staining process is in a sterile state.

[0073] Then, the cell solution and fluorescent staining solution are added to different feed cylinders 2 respectively. At this time, the forward and reverse motor 54 is started. The forward and reverse motor 54 drives the extrusion plates 51 on both sides of the feed cylinder 2 to move relative to each other through the threaded rod 53. The extrusion plate 51 and the execution plate 511 apply extrusion force to the flexible tube 22, causing the flexible tube 22 to deform. This controls the flow rate of the cell solution and fluorescent staining solution inside the flexible tube 22 so that they can be quantitatively added to the mixing cylinder 3.

[0074] S2: Cell Mixed Staining: The positioning motor 632 drives gear 1 633 and gear 2 634 to rotate clockwise synchronously. Gear 1 633 drives the ring gear 621 to rotate clockwise via idler wheel 631. The ring gear 621 drives the rotating plate 625, smoothing plate 626 and actuating block 628 to rotate clockwise synchronously via support rod 622. At the same time, gear 2 634 drives the storage tank 61 to rotate counterclockwise via positioning gear 635. The storage tank 61 drives the cell fluid and fluorescent staining solution at its bottom to rotate counterclockwise. Thus, the smoothing plate 626 and actuating block 628 rotate in opposite directions with the storage tank 61, so as to enhance the mixing intensity of the cell fluid and fluorescent staining solution by the smoothing plate 626 and actuating block 628.

[0075] During this process, the agitator 628 can cut a slit in the cell sap, allowing the fluorescent staining solution on the surface of the cell sap to enter the slit and stain the cells on the inner wall of the slit. Then, the smoothing plate 626 smooths the slit, restoring the cell sap and fluorescent staining solution to their original state. At this time, the fluorescent staining solution in the slit is squeezed into the cell sap. By repeating this step, the cell sap and fluorescent staining solution can be repeatedly cut and smoothed, thereby ensuring thorough mixing of the cell sap and fluorescent staining solution and improving the overall staining of the cell sap.

[0076] S3: Quantitative removal of stained cells: Repeatedly pushing and releasing the pressing plate 642 causes the pressing plate 642 to drive the telescopic bellows 641 to repeatedly extend and retract. Thus, the telescopic bellows 641 draws out the stained cell fluid from the storage tank 61 into the telescopic bellows 641 through the first one-way valve 644. Then, the cell fluid inside is squeezed out into the external flow tube through the second one-way valve 645. This completes the discharge of cell fluid.

[0077] In addition, the position of the nut 648 can be adjusted by rotating it; when the pressing plate 642 is pushed, the pressing plate 642 abuts against the nut 648, thereby controlling the moving distance of the pressing plate 642, and thus controlling the squeezing amplitude of the telescopic bellows 641, thereby controlling the quantitative discharge of cell fluid in the telescopic bellows 641, which is convenient to operate.

[0078] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0079] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A fluorescence staining apparatus for flow cytometry analysis, comprising a worktable, two feed cylinders disposed on the worktable, a mixing cylinder mounted at the lower end of the worktable, and a nitrogen filling machine disposed at the upper end of the worktable, the output end of the nitrogen filling machine extending into the mixing cylinder, characterized in that: The upper part of the worktable is equipped with a quantitative feeding component that works in conjunction with the feed cylinder. The quantitative feeding component includes two extrusion plates located on both sides of the feed cylinder. A cell mixing component is installed inside the mixing cylinder, wherein: The cell mixing component includes a storage tank rotatably connected to the lower end of the mixing cylinder. A mixing assembly is provided on the inner wall of the mixing cylinder. The mixing assembly includes an annular toothed ring rotatably connected to the lower side of the inner wall of the mixing cylinder. A driving assembly is installed between the mixing cylinder and the storage tank. The driving assembly includes an idler wheel disposed inside the mixing cylinder and meshing with the annular toothed ring. A discharge assembly is provided on the outer wall of the storage tank near its lower side. The discharge assembly includes a telescopic corrugated pipe. The quantitative feeding component also includes vertical plates. Two vertical plates are symmetrically arranged at the upper end of the worktable along its length direction. Two threaded rods are symmetrically rotatably connected between the vertical plates along the width direction of the worktable. The two threaded rods are connected by a belt drive. A forward and reverse motor connected to the threaded rod is provided on any vertical plate through a motor cover. Two threads with opposite directions are symmetrically opened on the outer wall of the threaded rod along the feed cylinder. An adjustment limit component is provided between the threaded rod and the extrusion plate. The mixing component also includes a support rod. The inner wall of the annular toothed ring is provided with a support rod. The lower end of the support rod is equipped with a rotating plate through two support rods symmetrically arranged along its length. The outer wall of the support rod is fitted with a scraper that slides in contact with the inner wall of the storage tank. A smoothing plate is installed in the middle of the lower end of the rotating plate, and two cutting groups are symmetrically arranged along the smoothing plate at the lower end of the rotating plate. The lower end of the smoothing plate is in rotatable contact with the bottom wall of the storage tank. Both sides of the smoothing plate in the width direction are provided with chamfers, and the side of the chamfer away from the middle of the smoothing plate gradually slopes upward. Each cutting group includes multiple actuating blocks arranged at equal intervals along the length of the rotating plate. The lower end of the actuating block slides in contact with the bottom wall of the storage tank. The actuating block is a triangular pyramid structure used to cut the cell fluid and staining solution. The multiple actuating blocks in the same cutting group are divided into two groups with the middle of the rotating plate as the center. The two groups of actuating blocks are arranged in a centrally symmetrical manner along the middle of the rotating plate, and the actuating blocks of the two cutting groups are arranged alternately. The drive assembly also includes a positioning motor. The positioning motor is mounted on the outer wall of the mixing drum via a motor mount. The output shaft of the positioning motor is provided with a first gear and a second gear from top to bottom. The first gear meshes with an idler gear. The outer wall of the storage drum is fitted with a positioning gear ring that meshes with the second gear.

2. The fluorescence staining apparatus for flow cytometry analysis according to claim 1, characterized in that: The adjusting and limiting component includes a rectangular sleeve. Multiple rectangular sleeves are fitted onto the outer wall of the threaded rod by a threaded connection. The rectangular sleeves slide through the extrusion plate. Multiple limiting holes are equally spaced along the axis of the threaded rod on the side of the rectangular sleeves near the middle of the extrusion plate. A receiving groove is provided inside the extrusion plate. A pressure rod is slidably connected inside the receiving groove. A fixed magnetic block is installed on the bottom wall of the receiving groove. A displacement magnetic block that cooperates with the fixed magnetic block is provided at the lower end of the pressure rod. Triangular blocks are installed on both sides of the pressure rod relative to the rectangular sleeve. The inclined surface of the triangular blocks gradually slopes towards the middle of the pressure rod from top to bottom. Two circular holes are symmetrically opened inside the extrusion plate along the receiving groove. An actuating contact rod is slidably connected in the circular holes. One end of the actuating contact rod slides and abuts against the inclined surface of the triangular block, and the other end is installed with a clamping pin through the connecting plate. The end of the clamping pin away from the connecting plate passes through the extrusion plate and cooperates with the limiting hole. A retraction spring sleeved on the outside of the actuating contact rod is installed between the side of the connecting plate away from the clamping pin and the inner wall of the circular hole.

3. The fluorescence staining apparatus for flow cytometry analysis according to claim 1, characterized in that: The discharge assembly also includes a pressing plate sleeved on the outer wall of the telescopic corrugated pipe on the side away from the storage tank. A top extension spring sleeved on the outside of the telescopic corrugated pipe is installed between the pressing plate and the outer wall of the storage tank. A first one-way valve is installed inside the telescopic corrugated pipe on the side near the storage tank, and a second one-way valve is installed inside the telescopic corrugated pipe on the side away from the storage tank. A baffle is provided between the storage tank and the pressing plate.

4. The fluorescence staining apparatus for flow cytometry analysis according to claim 3, characterized in that: The partition includes threaded guide rods. Multiple threaded guide rods are evenly arranged in annular patterns along the telescopic corrugated pipe on the outer wall of the storage tank. The threaded guide rods slide through the pressing plate. Each threaded guide rod has a nut fitted on its outer wall by means of threaded connection. Multiple nuts are connected to each other by a belt.

5. The fluorescence staining apparatus for flow cytometry analysis according to claim 1, characterized in that: The upper end of the feed cylinder is provided with a sealing ring to prevent oxygen from entering. The upper half of the feed cylinder is symmetrically provided with two supports connected to the worktable along its axis. The lower half of the feed cylinder extends into the mixing cylinder after passing through the worktable. The middle part of the feed cylinder is a flexible tube that is easy to deform. An execution plate that abuts against the flexible tube is installed on the side of the extrusion plate near the feed cylinder.

6. A fluorescence staining method for flow cytometry analysis, comprising a fluorescence staining apparatus for flow cytometry analysis as described in any one of claims 1-5, characterized in that, The fluorescent staining method includes the following steps: S1: Adding cell slurry and staining solution: First, nitrogen gas is added into the device through a nitrogen filling machine. Then, cell slurry and staining solution are added into the mixing cylinder in a certain proportion through a quantitative feeding component. S2: Cell Mixing and Staining: The cell mixture and staining solution in S1 are mixed evenly through the cell mixing component; S3: Quantitative Removal of Stained Cells: The stained cells are quantitatively removed according to the needs of detection and analysis through the discharge component.

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