Flow cytometry cell pretreatment detection tube, device and method

Abstract

The invention provides a flow cytometry cell pretreatment detection tube, device and method.The flow cytometry cell pretreatment detection tube comprises a sample loader and an integrated filtering and antibody pre-coating negative pressure detection tube, the integrated filtering and antibody pre-coating negative pressure detection tube comprises a negative pressure detection tube head and a negative pressure detection tube body, and the sample loader is in destructive connection with an upper inlet of the negative pressure detection tube head; a cell filter screen is arranged in the negative pressure detection tube head, a bottom outlet of the negative pressure detection tube head is hermetically and detachably connected with an opening of the negative pressure detection tube body, and a pre-coating area is arranged at the bottom of the negative pressure detection tube body and is used for bearing a marker. The technical problems that in the prior art, when a single-cell suspension is prepared, the device structure and operation are complex, the manufacturing cost is high, and integrated and rapid fluorescent antibody incubation cannot be achieved at the same time are solved.

Description

Technical Field

[0001] The invention belongs to the technical field of clinical detection and scientific research, and specifically belongs to a flow cytometry cell pre-treatment detection tube, device and method. Background Art

[0002] Flow cytometry (FCM) is a technique used for quantitative cell analysis, which can perform multi-parameter, rapid quantitative analysis of single cells or other biological particles at the cellular molecular level. It is currently widely used in clinical medicine, basic medicine, and biological-related clinical testing and scientific research fields.

[0003] Flow cytometry requires the use of a flow cytometer for detection and analysis, which consists of a fluid system, an optical system, and an electronic system. Its working principle is to allow dispersed, fluorescently labeled single cells or particles suspended in a liquid to pass through the detection area one by one, while the fluorescence detector captures the fluorescence signal and converts it into electrical pulse signals representing the forward scattering angle, side scattering angle, and different fluorescence intensities. These signals can be processed by a computer to form corresponding scatter plots, histograms, density maps, and three-dimensional structural images, etc., for analysis.

[0004] When the flow cytometer is working, it needs to make single cells or particles pass through the detection area one by one and be detected. Therefore, the samples used for flow cytometry are usually single cell suspensions, which can come from blood, body fluids, cultured cells or tissue cells, etc. When preparing samples, it is necessary to shorten the time required for the cell processing process to ensure cell activity as much as possible, and at the same time ensure that the cell suspension is a single cell suspension to meet the needs of subsequent experiments.

[0005] The working principle of flow cytometer is to capture fluorescent signals through fluorescent detectors. Before detection, cells need to be labeled with fluorescent antibodies or fluorescent dyes. Fluorescent antibodies are produced by covalently binding monoclonal or polyclonal antibodies that specifically recognize a certain antigen index to fluorescein. This combination does not affect the antibody's specific recognition of the antigen, and enables the antibody to obtain the optical properties of fluorescein. Therefore, the prepared single cell suspension needs to be incubated in a constant temperature and away from light using specific fluorescent antibodies, so as to fluorescently label cells that specifically express a certain antigen, and convert them into data results that can be used for analysis through flow cytometer detection. Fluorescent dyes can directly bind to DNA in cells and develop color.

[0006] However, in the existing technology, the single-cell suspension preparation device generally has a complex structure and operation, and the device cost is relatively high. The existing device takes a long time to process cells, and the process is complicated, which often causes irreversible damage to the activity state of the cells. At the same time, the existing devices generally only have the function of preparing single-cell suspensions, and cannot achieve integrated and rapid fluorescent antibody incubation, making it difficult to ensure the consistency of cell activity, fluorescent antibody incubation, and repeatability of experimental results. Summary of the invention

[0007] In order to solve the problems existing in the prior art, the present invention provides a flow cytometry cell pretreatment detection tube, device and method to solve the technical problems in the prior art of preparing single cell suspension, such as complex device structure and operation, high cost, and inability to simultaneously achieve integrated and rapid fluorescent antibody incubation.

[0008] To achieve the above-mentioned purpose, the present invention provides the following technical solutions: a flow cytometry cell pretreatment detection tube, comprising a sample loader and an integrated filtration and antibody pre-coated negative pressure detection tube, the integrated filtration and antibody pre-coated negative pressure detection tube comprising a negative pressure detection tube head and a negative pressure detection tube body, the sample loader is destructively connected to the upper inlet of the negative pressure detection tube head, a cell filtration screen is arranged in the negative pressure detection tube head, the bottom outlet of the negative pressure detection tube head is sealed and detachably connected to the opening of the negative pressure detection tube body, a pre-coated area is arranged at the bottom of the negative pressure detection tube body, and the pre-coated area is used to carry a marker.

[0009] Furthermore, the sampler includes a sampler cavity, a sampler cavity cover, a sampler cavity protective cover, and an injection needle. The top of the sampler cavity is connected to a detachable sampler cavity cover, an injection needle is arranged at the bottom outlet of the sampler cavity and is connected to the injection needle, the sampler cavity protective cover is arranged on the outside of the sampler cavity, the top of the sampler cavity is detachably fixedly connected to the sampler cavity, and the bottom is used to be mounted on the head of the negative pressure detection tube, and the injection needle is arranged in the sampler cavity protective cover.

[0010] Furthermore, a rubber sealing cover is provided at the inlet of the negative pressure detection tube head, and the injection needle of the sampler penetrates the rubber sealing cover and is connected to the negative pressure detection tube head; the distance between the cell filtering screen in the negative pressure detection tube head and the rubber sealing cover is greater than the length of the injection needle.

[0011] Furthermore, the aperture of the cell filtration mesh is set according to the requirements of the cell sample; and the marker is one or more fluorescent antibodies or fluorescent dyes used in flow cytometry detection.

[0012] Furthermore, it also includes a sampler bracket and a light-proof support bracket. Multiple samplers are placed on the sampler bracket, and multiple negative pressure detection tubes are placed on the light-proof support bracket. The sampler bracket is placed on the light-proof support bracket to match the positions of the samplers and the negative pressure detection tubes. After pressing downward, the samplers are destructively connected to the upper inlet of the negative pressure detection tube heads in batches.

[0013] The present invention also provides an integrated rapid cell pretreatment device, including an intelligent temperature-controlled shaking light-proof incubator and the above-mentioned flow cytometry cell pretreatment detection tube, wherein the integrated filtration and antibody pre-coated negative pressure detection tube is placed on a light-proof support frame and sent into the intelligent temperature-controlled shaking light-proof incubator for cell pretreatment.

[0014] Furthermore, the intelligent temperature-controlled oscillating light-proof incubator includes an incubator body, a cover is arranged on the top of the incubator body, a cavity is arranged inside the incubator body for placing a light-proof support frame, and the incubator body has built-in self-test module, timing module, temperature setting module and oscillation module, wherein: the self-test module is used to detect whether the integrated filter and antibody pre-coated negative pressure detection tube placed on the light-proof support frame is installed in place; the timing module is used to realize the cell pre-treatment time setting; the temperature setting module is used to reach and maintain the set temperature during cell pre-treatment; the oscillation module is used to realize oscillation during cell pre-treatment.

[0015] Furthermore, the self-test module includes a self-test switch, a self-test indicator light, a through-beam photoelectric sensor receiver and a through-beam photoelectric sensor transmitter. The through-beam photoelectric sensor receiver and the through-beam photoelectric sensor transmitter are arranged on opposite sides of the cavity. The through-beam photoelectric sensor receiver and the through-beam photoelectric sensor transmitter are both connected to the power module through the self-test switch. The self-test indicator light is connected to the through-beam photoelectric sensor receiver to prompt that the integrated filtration and antibody pre-coated negative pressure detection tube is installed in place.

[0016] Furthermore, the timing module includes a timing setting button and a timer for alternating cycles with a set number of times, and the timer for alternating cycles with a set number of times is connected to the power module via the timing setting button; The oscillation module includes an oscillation switch, a DC motor speed regulator, and a DC motor with a reducer. The oscillation switch is connected to a fixed number of alternating cycle timer and a DC motor speed regulator. The DC motor speed regulator and the DC motor with a reducer are connected to a power module. The temperature setting module includes a temperature setting button, a temperature control probe, a precision constant temperature controller and a heater. The temperature control probe is connected to the precision constant temperature controller, the precision constant temperature controller is connected to the heater, the temperature control probe, the heater and the precision constant temperature controller are all connected to the power module, the temperature setting button is used to set the temperature setting value of the precision constant temperature controller; the temperature control probe is used to detect the current temperature and feed back to the precision constant temperature controller; when the precision constant temperature controller receives that the temperature is lower than the set value, it outputs a voltage to the heater to achieve heating.

[0017] The present invention also provides a flow cytometry detection method, which is implemented using the above-mentioned integrated rapid cell pre-treatment device, as follows: The cell suspension is added to the sampler. After the sampler is destructively connected to the upper inlet of the negative pressure detection tube head, the cell suspension is sucked into the negative pressure detection tube head under the action of negative pressure. The cell suspension is filtered through the cell filter mesh in the negative pressure detection tube head to obtain a single cell suspension. The single cell suspension further enters the negative pressure detection tube body under the action of negative pressure and mixes with the marker in the pre-coated area to obtain a labeled single cell suspension; The integrated filtration and antibody pre-coated negative pressure detection tube containing the labeled single cell suspension is placed in a light-proof support frame, and the light-proof support frame is placed in an intelligent temperature-controlled oscillating light-proof incubator. The intelligent temperature-controlled oscillating light-proof incubator starts the self-checking module, and after detecting that the integrated filtration and antibody pre-coated negative pressure detection tube placed on the light-proof support frame is installed in place, the intelligent temperature-controlled oscillating light-proof incubator starts the timing module, the temperature setting module and the oscillation module to perform timed heat preservation and oscillation light-proof incubation on the labeled single cell suspension; After the incubation is completed, the negative pressure detection tube head and the sample loader are removed from the negative pressure detection tube body, and the negative pressure detection tube body is sent to the flow cytometer for flow detection.

[0018] Compared with the prior art, the present invention has at least the following beneficial effects: The integrated filtration and antibody pre-coated negative pressure detection tube provided by the present invention realizes the rapid filtration and single cellization of cell suspension. Through the destructive connection between the sampler and the negative pressure detection tube head, the cell suspension is quickly sucked into the detection tube under the drive of negative pressure and passes through a precise cell filtration screen. In this process, the cells are effectively dispersed into a single state, and due to the effect of negative pressure, the filtration efficiency is significantly improved, which greatly shortens the preparation time; and the integrated filtration and antibody pre-coated negative pressure detection tube also integrates the function of marker pre-coating. Traditional fluorescent antibody incubation usually requires separate steps and containers, which is not only cumbersome to operate, but also easy to introduce pollution. The present invention pre-carries one or more fluorescent antibodies or fluorescent dyes required for flow cytometry detection by setting a pre-coated area at the bottom of the negative pressure detection tube. When the cell suspension passes through the filter screen, it is directly mixed with the pre-coated marker, thereby realizing the rapid combination of cells and markers. This design not only simplifies the experimental steps and reduces the risk of contamination, but also improves the incubation efficiency, making the fluorescent labeling process more stable and reliable.

[0019] Furthermore, the detection tube of the present invention has good sealing and detachability. The sealing connection between the negative pressure detection tube head and the tube body ensures the negative pressure stability and sterile environment during the detection process. At the same time, the detachable design of the tube head and the tube body makes it easy to remove the tube head after the incubation is completed, and directly perform flow cytometer detection without additional transfer steps, further improving the experimental efficiency and accuracy.

[0020] Furthermore, the pore size of the cell filtration screen of the present invention can be flexibly selected according to the size of different cell types, thereby ensuring the filtration effect while minimizing the physical damage to the cells.

[0021] Furthermore, the sizes and numbers of the sample loader bracket and the light-shielding support frame of the present invention match each other, so batch transfer and sample loading can be achieved, which greatly improves the operation efficiency and shortens the operation time.

[0022] The integrated rapid cell pretreatment device of the present invention integrates multiple functions such as single cell suspension preparation, fluorescent antibody incubation, cell constant temperature, oscillation and light-proof incubation, realizing the integrated operation of cell pretreatment. The user only needs to put the integrated filtration and antibody pre-coated negative pressure detection tube into the device to automatically complete the whole process from cell suspension preparation to fluorescent antibody incubation. This design not only simplifies the experimental steps and reduces the difficulty of operation, but also greatly improves the experimental efficiency and accuracy.

[0023] The device of the present invention adopts intelligent temperature-controlled oscillation light-proof incubation technology to ensure the best reaction conditions of cells during the incubation process. Through the built-in self-check module, timing module, temperature setting module and oscillation module, the device can accurately control the incubation time, temperature and oscillation frequency, thereby meeting the condition requirements of different types of experiments. At the same time, the light-proof treatment also effectively prevents the decomposition of photosensitive substances, ensuring the reliability of the experimental results.

[0024] In addition, the device is highly flexible and scalable. Users can choose different cell filter screens, fluorescent antibodies, light-proof support frames and other accessories according to experimental requirements, so as to achieve rapid pre-treatment of different types of cells. At the same time, the device can also be seamlessly connected with other experimental equipment (such as flow cytometers), realizing full automation from cell pre-treatment to data analysis.

[0025] The present invention also provides a flow cytometry detection method based on an integrated rapid cell pretreatment device, which realizes the rapid preparation of cell suspension and efficient incubation of fluorescent antibodies through the integrated rapid cell pretreatment device. Compared with the traditional preparation method, this method not only simplifies the experimental steps, reduces the operation time, but also reduces the risk of contamination and cell damage. At the same time, the efficient incubation of fluorescent antibodies also makes cell labeling more stable and reliable, and improves the sensitivity, accuracy and repeatability of the detection. The selected intelligent temperature control oscillation light-proof incubation technology ensures the best growth environment for cells during the incubation process. By accurately controlling parameters such as incubation time, temperature and oscillation frequency, the combination of cells and fluorescent markers can be effectively promoted to improve the labeling efficiency. At the same time, the light-proof treatment also effectively prevents the decomposition of photosensitive substances, ensuring the reliability of the test results. Through the seamless connection between the integrated rapid cell pretreatment device and the flow cytometer, the whole process from cell pretreatment to data analysis is realized. The user only needs to simply set the experimental parameters and start the device to automatically complete the steps of preparation of cell suspension, incubation of fluorescent antibodies, cell insulation, oscillation and light-proof treatment, and finally obtain accurate test results. This design not only greatly improves experimental efficiency, but also reduces operational difficulty and human errors, providing scientific researchers with a more convenient and efficient experimental method. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the structural diagram of the intelligent temperature-controlled oscillating light-proof incubator; Figure 2 This is a schematic diagram of the use of the intelligent temperature-controlled oscillating light-proof incubator; Figure 3 This is a structural breakdown diagram of the intelligent temperature-controlled oscillating light-proof incubator; Figure 4 This is the structural diagram of the integrated filtration and antibody pre-coated negative pressure detection tube; Figure 5 This is a structural diagram of the sampler cavity protection cover; Figure 6 is a structural diagram of the sample loader bracket; Figure 7 This is a circuit diagram of the intelligent temperature-controlled oscillating light-proof incubator of the present invention; In the attached figure: 1. Intelligent temperature-controlled oscillating light-proof incubator; 2. Incubator body; 3. Machine cover; 4. Light-proof support frame; 5. Power socket; 6. Self-test indicator light; 7. Opposing photoelectric sensor receiver; 8. Opposing photoelectric sensor transmitter; 9. Main switch; 10. Self-test switch; 11. Timing setting button; 12. Temperature setting button; 13. Oscillation switch; 14. Control panel; 15. Integrated filtration and antibody pre-coated negative pressure detection tube; 16. Sampler cavity cover; 17. Minimum sample loading line; 18. Sampler; 19. Sampler cavity protection cover; 20. Injection needle; 21. Negative pressure detection Tube head; 22. Cell filter screen; 23. Negative pressure detection tube body; 24. Pre-coated area; 25. External thread; 26. Internal thread; 27. Rubber sealing cover; 28. Needle sleeve; 29. ​​Minimum detection line; 30. Sealing rubber ring; 31. Sample loader bracket; 32. Main power switch; 33. Detection switch; 34. Timer switch; 35. Current limiting resistor; 36. Temperature control probe; 37. Heater; 38. LED indicator; 39. Power module; 40. Precision constant temperature controller; 41. Alternating cycle timer with a set number of times; 42. DC motor speed regulator; 43. DC motor with reducer. DETAILED DESCRIPTION

[0027] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0028] The present invention provides an integrated rapid cell pre-treatment device, which is mainly used for sample pre-treatment of cells from various sources before flow cytometer detection, including an intelligent temperature-controlled oscillating light-proof incubator 1, an integrated filtering and antibody pre-coated negative pressure detection tube 15, wherein: like Figures 1 to 3As shown, the intelligent temperature-controlled oscillating light-proof incubator 1 includes an incubator body 2, a machine cover 3 is arranged on the top of the incubator body 2, a cavity is arranged inside the incubator body 2 for placing a light-proof support frame 4, the light-proof support frame 4 is used to place an integrated filtering and antibody pre-coated negative pressure detection tube 15, and opposite sides of the cavity are respectively provided with a through-type photoelectric sensor receiver 7 and a through-type photoelectric sensor transmitter 8, and the installation height is flush with the top of the light-proof support frame 4; the incubator body 2 can realize the functions of self-checking, timing, temperature setting, and oscillation, and the control screen 14 of the incubator body 2 is provided with a main switch 9, a self-check switch 10, a timing setting button 11, a temperature setting button 12, and an oscillation switch 13. The intelligent temperature-controlled oscillating light-proof incubator 1 is connected to the power socket 5 to realize light-proof, constant temperature, timing and oscillation incubation of single cell suspension and fluorescent antibodies or dyes.

[0029] The self-test module includes a self-test switch 10, a self-test indicator light 6, a through-beam photoelectric sensor receiver 7 and a through-beam photoelectric sensor transmitter 8. The through-beam photoelectric sensor receiver 7 and the through-beam photoelectric sensor transmitter 8 are arranged opposite to each other. The through-beam photoelectric sensor receiver 7 and the through-beam photoelectric sensor transmitter 8 are both connected to the 220V / 12V power supply module 39 through the self-test switch 10, and are used to detect whether the integrated filter and antibody pre-coated negative pressure detection tube 15 placed on the light-proof support frame 4 is installed in place. The self-test indicator light 6 is connected to the through-beam photoelectric sensor receiver 7. When the detection is appropriate, the self-test indicator light 6 lights up for prompting.

[0030] The timing module includes a timing setting button 11 and a timer 41 for alternating cycles with a set number of times. The timer 41 for alternating cycles with a set number of times ... The temperature setting module includes a temperature setting button 12, a temperature control probe 36, a precision constant temperature controller 40 and a heater 37. The temperature control probe 36 is connected to the precision constant temperature controller 40, which is connected to the heater 37. The temperature control probe 36, the heater 37 and the precision constant temperature controller 40 are connected to a 220V / 12V power supply module 39. The temperature setting value of the precision constant temperature controller 40 is set by the temperature setting button 12. The temperature control probe 36 detects the current temperature and feeds back to the precision constant temperature controller 40. When the temperature is lower than the set value, the precision constant temperature controller 40 outputs a 12V voltage to the heater 37 to start heating, thereby realizing the heating of the intelligent temperature-controlled oscillating light-proof incubator.

[0031] The oscillation module includes an oscillation switch 13, a timer 41 for alternating cycles with a set number of times, a DC motor speed regulator 42, and a DC motor with a reducer 43. The oscillation switch 13 is connected to the timer 41 for alternating cycles with a set number of times, and is connected to the DC motor speed regulator 42, and the DC motor with a reducer 43 is connected to the 220V / 12V power supply module 39. The oscillation switch 13 is set to set the timer 41 for alternating cycles with a set number of times, and the oscillation function of the intelligent temperature-controlled oscillating light-shielding incubator is realized through the DC motor speed regulator 42 and the DC motor with a reducer 43.

[0032] The light-proof support frame 4 is used to load the integrated filtration and antibody pre-coated negative pressure detection tube 15, and the common specification is 4×3, which can also be replaced according to experimental requirements. After the integrated filtration and antibody pre-coated negative pressure detection tube 15 after sample addition is inserted into the light-proof support frame 4, the subsequent incubation steps can be carried out through the intelligent temperature control oscillation light-proof incubator 1.

[0033] like Figures 4 to 6 As shown, the integrated filtering and antibody pre-coated negative pressure detection tube 15 includes a sampler 18, a negative pressure detection tube head 21 and a negative pressure detection tube 23, wherein: The sampler 18 includes a sampler cavity, a sampler cavity cover 16, a minimum sample loading line 17, a sampler cavity protective cover 19, an injection needle 20, and a needle sleeve 28. The top of the sampler cavity is connected to the detachable sampler cavity cover 16, the side wall of the sampler cavity is provided with the minimum sample loading line 17, the injection needle 20 is provided at the bottom outlet of the sampler cavity and is connected with the injection needle 20, and the needle sleeve 28 is used to be sleeved on the injection needle 20; the sampler cavity protective cover 19 is arranged on the outside of the sampler cavity, and its top is fixedly connected to the sampler cavity, and is a detachable device, and its bottom is used to be sleeved on the negative pressure detection tube body 23. The injection needle 20 is arranged in the sampler cavity protective cover 19, which can avoid the risk of puncture and facilitate the recovery of sharp waste.

[0034] The negative pressure detection tube includes a negative pressure detection tube head 21 and a negative pressure detection tube body 23, wherein the negative pressure detection tube head 21 is destructively connected to the sampler 18 after the needle cover 28 is removed, and the negative pressure detection tube head 21 and the negative pressure detection tube body 23 are connected to each other through a connection port with a sealing rubber ring and a thread; A rubber sealing cover 27 is provided at the inlet of the negative pressure detection tube head 21, and the injection needle 20 of the sampler 18 can penetrate the rubber sealing cover 27 and communicate with the negative pressure detection tube head 21; a layer of cell filter screen 22 is provided in the negative pressure detection tube head 21, and the distance between the cell filter screen 22 and the rubber sealing cover 27 is greater than the length of the injection needle 20; the outlet of the negative pressure detection tube head 21 is provided with an external thread 25 and a sealing rubber ring 30; An internal thread 26 is provided at the inlet of the negative pressure detection tube body 23, and the internal thread 26 matches the external thread 25 of the negative pressure detection tube head 21. A pre-coated area 24 is provided at the bottom of the negative pressure detection tube body 23, and the pre-coated area 24 is used to carry a marker; a minimum detection line 29 is provided on the negative pressure detection tube body 23; When performing cell pretreatment, the cell suspension is added to the sampler cavity of the sampler 18, and the liquid volume needs to exceed the minimum sample loading line 17. After the sampler cavity cover 16 is tightly closed, the needle cover 28 is removed, and the injection needle is pierced through the rubber sealing cover 27 of the negative pressure detection tube head 21. Then, the cell suspension is sucked into the negative pressure detection tube head 21 under the action of negative pressure, and the cells are filtered through the cell filter screen 22 therein to obtain a single cell suspension; the single cell suspension is sucked into the negative pressure detection tube body 23 under the action of negative pressure and mixed with the marker in the pre-coated area 24.

[0035] Preferably, the marker is one or more fluorescent antibodies or fluorescent dyes used in flow cytometry detection, and the pre-coated fluorescent antibodies or fluorescent dyes can be selected according to the experimental requirements, such as CD24, CD44, CD133, PI, DAPI, etc. The present invention uses a detection tube pre-coated with antibodies, which eliminates the steps of transferring cell suspensions, adding antibodies, mixing, etc., greatly reducing the experimental steps and repetitive manual operations. The process of adding micropipettes of antibodies is avoided, the systematic error between different samples is minimized, and the repeatability of the same sample during detection is greatly improved, thereby maximizing the accuracy of the detection. At the same time, rapid and high-throughput incubation of fluorescent antibodies or dyes is achieved, greatly improving efficiency.

[0036] Preferably, multiple sample loaders 18 are placed on the sample loader bracket 31, and multiple integrated filtration and antibody pre-coated negative pressure detection tubes 15 are placed on the light-proof support frame 4, and the positions of the sample loaders 18 and the integrated filtration and antibody pre-coated negative pressure detection tubes 15 match; when operating multiple different cell samples at the same time, multiple sample loaders 18 are placed on the sample loader bracket 31, and multiple negative pressure detection tubes are placed on the light-proof support frame 4, and the positions of the sample loaders 18 and the integrated filtration and antibody pre-coated negative pressure detection tubes 15 match. After removing the needle sleeve 28, the sample loader bracket 31 is placed on the light-proof support frame 4 and pressed downward to achieve high-throughput rapid loading and improve operating efficiency.

[0037] Preferably, the cell filter mesh 22 can be selected with different pore sizes and specifications such as 40 μm, 70 μm, 100 μm, etc. according to experimental requirements, so as to obtain a single cell suspension in the shortest time and in a manner that minimizes cell damage.

[0038] After the cell sample is sucked into the integrated filtration and antibody pre-coated negative pressure detection tube 15 under the action of negative pressure through the sample loader 18, the light-proof support frame 4 is immediately moved into the cavity of the intelligent temperature-controlled oscillating light-proof incubator, and the main switch 9 is turned on. After turning on the self-test switch 10, the self-test indicator light 6 prompts that the self-test has passed after the detection of the opposing photoelectric sensor confirms that the integrated filtration and antibody pre-coated negative pressure detection tube 15 is placed correctly. According to the requirements of the experimental conditions, the temperature, timing and oscillation switches are used to achieve light-proof, constant temperature, timing and oscillation incubation of single cell suspension and fluorescent antibodies or dyes. The use of the intelligent temperature-controlled oscillating light-proof incubator ensures the integrated rapid cell pre-treatment before flow cytometry detection under the conditions of high efficiency, constant experimental conditions, high throughput and reduced operating steps.

[0039] By using an integrated filtration and antibody pre-coated negative pressure detection tube and an intelligent temperature-controlled oscillating light-proof incubator, rapid incubation of fluorescent antibodies or dyes is achieved. The negative pressure detection tube head 21 and the negative pressure detection tube body 23 are connected by threads and sealing rubber rings. After the incubation is completed, the negative pressure detection tube head 21 with the sampler 18 is removed, and the flow cytometer can be matched for flow detection. Complex operation steps such as sample transfer are reduced, the experimental efficiency is improved and the error is reduced.

[0040] The circuit diagram of the intelligent temperature-controlled oscillating light-proof incubator is as follows: Figure 7 As shown, where: Main power switch K1 32: Located at the starting point of the circuit diagram, it is used to control the power on and off of the entire device.

[0041] Power module 39: marked as "AC, 220V / 12V", responsible for converting the input 220V alternating current into 12V direct current for use by subsequent circuits.

[0042] Current limiting resistor R1 35: connected in series with LED indicator light D1 (self-test indicator light 6) to limit the current passing through D1 to prevent it from overheating and damage.

[0043] Temperature control probe R2 36: connected to the precision constant temperature controller 40, used to detect the current temperature and feed back to the controller.

[0044] Heater R3 37: controlled by a precision thermostat controller 40. When the temperature is lower than the set value, the thermostat outputs a 12V voltage to R3 to start heating, thereby realizing the heating of the intelligent temperature-controlled oscillating light-proof incubator.

[0045] Through-beam photoelectric sensor: It consists of a through-beam photoelectric sensor receiver 7 and a through-beam photoelectric sensor transmitter 8. When the test tube is at a normal height, the light emitted by the transmitter can be received by the receiver; when the test tube is at an abnormal height, the light is blocked and the receiver cannot receive the light.

[0046] LED indicator D1 38 (self-test indicator 6): When the photoelectric sensor detects that the test tube height is abnormal, D1 (self-test indicator 6) will light up to send out an alarm signal.

[0047] Detection switch K2 33 (self-test switch 10): controls the power supply of the photoelectric sensor. When K2 is closed, the photoelectric sensor starts working.

[0048] Timer switch K3 34 (timing setting button 11): used to set the alternating times and the rotation and stop time of the motor. The speed of the motor can be adjusted by adjusting the speed regulator potentiometer.

[0049] The DC motor speed regulator 42 and the DC motor with reducer 43 cooperate with the timer switch K3 34 (timing setting button 11) to realize the alternating rotation and stop of the motor, and are used to control the vibration of the light-shielding support frame.

[0050] The working process of the intelligent temperature-controlled oscillating light-proof incubator is as follows: 1. Turn on the main switch, the machine starts working, and the power module outputs DC 12V to supply each circuit; 2. DC 12V is first supplied to the precision thermostat controller 40. Before the set temperature is reached, the precision thermostat controller 40 outputs 12V voltage to supply the heater R3 to start heating. After the set temperature is reached, the precision thermostat controller 40 stops outputting and heating is suspended.

[0051] 3. After reaching the set temperature, open the cover, put in the test tube, turn on the detection switch K2, the photoelectric sensor is powered on and starts working, the through-beam photoelectric sensor transmitter 8 emits a light with a diameter of about 0.5 mm, which is directed to the through-beam photoelectric sensor receiver 7. When a test tube is higher than the specified range, the light is blocked by the upper part of the test tube, and the through-beam photoelectric sensor receiver 7 cannot receive the light. The receiving head outputs a high level, which is limited by the current limiting resistor R1, and the LED indicator D1 is lit to sound an alarm; at the same time, the light shines on the raised part of the test tube, and the test tubes higher than the specified range can be observed with the naked eye; when all test tubes are at normal height, each light is received by the corresponding receiving head, all receiving heads output low levels, and the LED indicator D1 is off, indicating normal.

[0052] 4. When there is no alarm on any circuit, turn off the detector switch K2 and close the cover; 5. Turn on the timer switch K3, set the number of alternations and the motor stop time respectively, adjust the DC motor speed regulator to the appropriate speed to achieve oscillation.

[0053] Example: The present invention takes the detection of the proportion of CD133 positive cells in the hepatocellular carcinoma cell line Huh7 cell line as an example, and the use steps are: The Huh7 cell line cultured in the culture dish was taken out from the incubator at 37°C and 5% CO2, digested with trypsin to form a cell suspension, centrifuged using a centrifuge and the supernatant discarded. The Huh7 cell sediment was resuspended into a Huh7 cell suspension using a balanced salt solution by vortexing.

[0054] The Huh7 cell suspension is transferred to the sampler 18. After checking that the cell suspension is above the minimum loading line 17, the sampler chamber cover 16 is tightly closed and removed from the sampler bracket 31. The needle cover 28 of the injection needle 20 is removed, and the injection needle 20 is used to pierce the rubber sealing cover 27 of the negative pressure detection tube head 21. At this time, under the action of negative pressure, the cell suspension passes through the cell filter mesh 22 with a pore size of 70 μm, forms a single cell suspension and flows into the negative pressure detection tube body 23. The bottom of the negative pressure detection tube body 23 is pre-coated with APC-CD133 fluorescent antibody. After checking that the liquid level of the single cell suspension is higher than the minimum detection line marked on the negative pressure detection tube body 23, the loading and single cell suspension pretreatment steps are completed.

[0055] Quickly move the integrated filtration and antibody pre-coated negative pressure test tube 15 containing the Huh7 cell line into the light-proof bracket 4 in the intelligent temperature-controlled oscillating light-proof incubator, and turn on the main switch and self-test switch. After the self-test indicator light indicates that the self-test has passed, quickly cover the machine cover, turn on the oscillation switch knob, oscillate and mix for 1 minute, set the reaction temperature to 37°C and set the reaction countdown to 30 minutes. After the countdown ends, turn on the oscillation again and oscillate and mix for 1 minute. The incubation step of the fluorescent antibody is completed.

[0056] The integrated filtration and antibody pre-coated negative pressure detection tube 15 is taken out from the intelligent temperature-controlled shaking light-proof incubator, the negative pressure detection tube head 21 is unscrewed, and it is directly connected to the flow cytometer to detect the fluorescence amount of APC in the Huh7 cell line. Through data processing and analysis, the detection result of the proportion of CD133 positive cells in the Huh7 cell line is obtained.

[0057] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the attached claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims are included in the present invention. Any figure mark in the claims should not be regarded as limiting the claims involved.

[0058] In addition, it should be understood that although this specification is described in accordance with the implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation modes that can be understood by those skilled in the art. The above content is only to illustrate the technical idea of ​​the present invention, and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution according to the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A flow cytometry cell pretreatment detection tube, characterized in that: The invention comprises a sampler (18) and an integrated filtering and antibody pre-coated negative pressure detection tube (15); the integrated filtering and antibody pre-coated negative pressure detection tube (15) comprises a negative pressure detection tube head (21) and a negative pressure detection tube body (23); the sampler (18) is destructively connected to the upper inlet of the negative pressure detection tube head (21); a cell filtering screen (22) is arranged in the negative pressure detection tube head (21); the bottom outlet of the negative pressure detection tube head (21) is sealed and detachably connected to the opening of the negative pressure detection tube body (23); a pre-coated area (24) is arranged at the bottom of the negative pressure detection tube body (23); the pre-coated area (24) is used to carry a marker.

2. A flow cytometry cell pretreatment detection tube according to claim 1, characterized in that: The sampler (18) comprises a sampler cavity, a sampler cavity cover (16), a sampler cavity protection cover (19), and an injection needle (20); the top of the sampler cavity is connected to a detachable sampler cavity cover (16); the injection needle (20) is arranged at the outlet of the bottom of the sampler cavity and is in communication with the injection needle (20); the sampler cavity protection cover (19) is arranged outside the sampler cavity, the top of which is detachably fixedly connected to the sampler cavity, and the bottom of which is used to be sleeved on a negative pressure detection tube head (21); and the injection needle (20) is arranged in the sampler cavity protection cover (19).

3. A flow cytometry cell pretreatment detection tube according to claim 2, characterized in that: A rubber sealing cover (27) is provided at the inlet of the negative pressure detection tube head (21); the injection needle (20) of the sampler (18) penetrates the rubber sealing cover (27) and is connected to the negative pressure detection tube head (21); the distance between the cell filtering screen (22) in the negative pressure detection tube head (21) and the rubber sealing cover (27) is greater than the length of the injection needle (20).

4. A flow cytometry cell pretreatment detection tube according to claim 1, characterized in that: The pore size of the cell filter screen (22) is set according to the requirements of the cell sample; the marker is one or more fluorescent antibodies or fluorescent dyes used in flow cytometry detection.

5. A flow cytometry cell pretreatment detection tube according to claim 1, characterized in that: It also includes a sampler bracket (31) and a light-shielding support bracket (4), wherein a plurality of samplers (18) are placed on the sampler bracket (31), and a plurality of negative pressure detection tubes are placed on the light-shielding support bracket (4). The sampler bracket (31) is placed on the light-shielding support bracket (4) so ​​that the positions of the samplers (18) and the negative pressure detection tubes match, and after pressing downward, the samplers (18) are destructively connected to the upper inlets of the negative pressure detection tube heads (21) in batches.

6. Integrated rapid cell pre-treatment device, characterized in that: The invention comprises an intelligent temperature-controlled shaking light-proof incubator (1) and a flow cytometry cell pre-treatment detection tube according to any one of claims 1 to 5, wherein the integrated filtering and antibody pre-coated negative pressure detection tube (15) is placed on a light-proof support frame (4) and sent into the intelligent temperature-controlled shaking light-proof incubator (1) for cell pre-treatment.

7. The integrated rapid cell pretreatment device according to claim 6, characterized in that: The intelligent temperature-controlled oscillating light-proof incubator (1) comprises an incubator body (2), a cover (3) is arranged on the top of the incubator body (2), a cavity is arranged inside the incubator body (2) for placing a light-proof support frame (4), and the incubator body (2) has a built-in self-check module, a timing module, a temperature setting module and an oscillation module, wherein: the self-check module is used to detect whether the integrated filter and antibody pre-coated negative pressure detection tube (15) placed on the light-proof support frame (4) is installed in place; the timing module is used to realize the cell pre-treatment time setting; the temperature setting module is used to reach and maintain the set temperature during the cell pre-treatment; and the oscillation module is used to realize oscillation during the cell pre-treatment.

8. The integrated rapid cell pretreatment device according to claim 7, characterized in that: The self-test module comprises a self-test switch (10), a self-test indicator light (6), a through-beam photoelectric sensor receiver (7) and a through-beam photoelectric sensor transmitter (8); the through-beam photoelectric sensor receiver (7) and the through-beam photoelectric sensor transmitter (8) are arranged facing each other on two opposite sides of the cavity; the through-beam photoelectric sensor receiver (7) and the through-beam photoelectric sensor transmitter (8) are both connected to the power module (39) via the self-test switch (10); the self-test indicator light (6) is connected to the through-beam photoelectric sensor receiver (7) to indicate that the integrated filtering and antibody pre-coated negative pressure detection tube (15) is installed in place.

9. The integrated rapid cell pretreatment device according to claim 7, characterized in that: The timing module comprises a timing setting button (11) and a timer (41) for alternating cycles with a set number of times; the timer (41) for alternating cycles with a set number of times is connected to the power module (39) via the timing setting button (11); The oscillation module comprises an oscillation switch (13), a DC motor speed regulator (42), and a DC motor with a reducer (43); the oscillation switch (13) is connected to a fixed-number alternating cycle timer (41) and the DC motor speed regulator (42); the DC motor speed regulator (42) and the DC motor with a reducer (43) are connected to a power module (39); The temperature setting module comprises a temperature setting button (12), a temperature control probe (36), a precision constant temperature controller (40) and a heater (37); the temperature control probe (36) is connected to the precision constant temperature controller (40), the precision constant temperature controller (40) is connected to the heater (37), the temperature control probe (36), the heater (37) and the precision constant temperature controller (40) are all connected to a power module (39); the temperature setting button (12) is used to set a temperature setting value of the precision constant temperature controller (40); the temperature control probe (36) is used to detect the current temperature and feed it back to the precision constant temperature controller (40); when the precision constant temperature controller (40) receives a temperature lower than the set value, it outputs a voltage to the heater (37) to achieve heating.

10. A flow cytometry detection method, characterized in that: The method is implemented by using the integrated rapid cell pretreatment device described in any one of claims 6 to 9, specifically as follows: The cell suspension is added to the sample loading device (18). After the sample loading device (18) is destructively connected to the upper inlet of the negative pressure detection tube head (21), the cell suspension is sucked into the negative pressure detection tube head (21) under the action of negative pressure. The cell suspension is filtered through the cell filter mesh (22) in the negative pressure detection tube head (21) to obtain a single cell suspension; The single cell suspension further enters the negative pressure detection tube body (23) under the action of negative pressure and mixes with the marker in the pre-coated area (24) to obtain a labeled single cell suspension; The integrated filtration and antibody pre-coated negative pressure detection tube (15) containing the labeled single cell suspension is placed in a light-proof support frame (4), and the light-proof support frame (4) is placed in an intelligent temperature-controlled oscillating light-proof incubator (1). The intelligent temperature-controlled oscillating light-proof incubator (1) starts a self-checking module. After detecting that the integrated filtration and antibody pre-coated negative pressure detection tube (15) placed on the light-proof support frame (4) is installed in place, the intelligent temperature-controlled oscillating light-proof incubator (1) starts a timing module, a temperature setting module and an oscillation module to perform timed heat preservation and oscillation light-proof incubation on the labeled single cell suspension; After the incubation is completed, the negative pressure detection tube head (21) and the sample loaders (18) are removed from the negative pressure detection tube body (23), and the negative pressure detection tube body (23) is sent to a flow cytometer for flow cytometry detection.

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