A method for cleaning the sample inlet of an automated wash type flow cytometer
By using Decon 90 cleaning agent and sodium hypochlorite solution for multiple cleaning cycles, the problem of detection error caused by residues at the sample inlet of the flow cytometer was solved, achieving higher instrument cleanliness and detection accuracy. This method is suitable for cleaning the sample inlet of flow cytometers.
Patent Information
- Application Number
- CN202410567929.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-05-09
- Publication Date
- 2026-02-24
- Estimated Expiration
- 2044-05-09
AI Technical Summary
Existing flow cytometers have residues at the sample inlet end when detecting low concentrations of microorganisms, which leads to discrepancies between the detection results and expected values. Furthermore, automatic cleaning methods cannot effectively remove biofilms and attachments at the sample inlet end, resulting in the detection of non-sample signals.
Decon 90 cleaning agent was used for pre-cleaning, formal cleaning, and re-cleaning. Decon 90 cleaning agent was used for pre-cleaning and re-cleaning, while sodium hypochlorite solution was used for formal cleaning. Residues at the injection end were removed through multiple cleanings, and the cleaning effect was monitored by data acquisition.
It significantly reduces stray signals and fluorescence values at the injection end, improves the cleanliness of the instrument, and ensures the accuracy and reliability of sample detection, especially reducing non-specific interference in the detection of low-concentration microorganisms.
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Figure CN118341733B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of instrument cleaning material, in particular to a method for cleaning the sample inlet end of an automatic cleaning type flow cytometer. BACKGROUND
[0002] Flow cytometers are widely used in toxicology, cell biology, immunology, and microbiology related fields, and have become an indispensable instrument for many research institutions. Analytical flow cytometers are mainly composed of four parts: flow chamber and liquid flow system; laser source and optical system; phototube and detection system; computer and analysis system. The flow chamber and liquid flow system are mainly responsible for restraining the cell or particle suspension sample through the sheath liquid, so that the cells or particles flow through the laser focused detection point one by one, generating multiple different parameter signals, which are collected by the detection system for subsequent analysis.
[0003] For the sample inlet components including the flow chamber, cleanliness is very important, and many flow cytometer manufacturers have established their own automatic cleaning programs to facilitate user use, such as Agilent Novocyte, BD FACSVia, Accuri C6Plus, ThermoFisher Attune NxT, Amnis FlowSight, SinoCyte, FlowCyte, etc.
[0004] Novocyte type flow cytometers are widely used in concentration quantification due to their sample volume parameter stop condition option. Whether it is a mammalian cell line, immune cells, or water or soil extracted microorganisms, cyanobacteria, or even bacteriophage viruses, there is a need to use Novocyte type flow cytometers to quantify the concentration of volume parameters. However, although the instrument administrator will perform automatic cleaning and flushing in time after each experiment according to the manufacturer's instructions, when performing low-concentration microbial quantification analysis of environmental water, the results will still not match the expected value and even be significantly different from the control group target value. In this case, even if the instrument is sampled with ultrapure water, a certain amount of particle count will still be detected. Based on this finding, it is speculated that this phenomenon is caused by residues in the sample inlet (including the sample inlet needle, sample inlet tube, and flow chamber), and the sample inlet may have adhered to the biological membrane, and each time the sample is sampled, some particles will be released with the sample liquid, thereby causing some non-sample signals to be detected during the test. SUMMARY
[0005] Therefore, the purpose of the present application is to provide a method for cleaning the sample inlet end of an automatic cleaning type flow cytometer. The sample inlet cleaning method provided by the present application can remove as much residue as possible to provide a relatively cleaner detection microenvironment for sample detection.
[0006] In order to achieve the above-mentioned purposes, the present application provides the following technical solutions.
[0007] The present application provides a kind of automatic cleaning type flow cytometer sample inlet cleaning method, comprising the following steps:
[0008] Automatic cleaning type flow cytometer is sequentially pre-cleaned, formally cleaned and re-cleaned;The cleaning fluid used in pre-cleaning and re-cleaning is Decon 90 cleaning agent;The cleaning fluid used in formal cleaning is sodium hypochlorite solution.
[0009] Preferably, the volume fraction of the Decon 90 cleaning agent is independently 5-10%.
[0010] Preferably, the number of pre-cleaning is 5 times.
[0011] Preferably, the number of re-cleaning is 5 times.
[0012] Preferably, the mass fraction of the sodium hypochlorite solution is 0.5-1.0% in terms of active chlorine.
[0013] Preferably, the number of formal cleaning is 15 times.
[0014] Preferably, the sodium hypochlorite solution is filtered through a 0.22 μm filter membrane before use.
[0015] Preferably, the sample inlet of each pre-cleaning, formal cleaning and re-cleaning is 100 μL, the sample flow rate is 66-120 μL / min, and the sample flow diameter is 16.8-22.7 μm.
[0016] Preferably, the time of pre-cleaning is 4.2-7.5 min, the time of formal cleaning is 12.5-22.5 min, and the time of re-cleaning is 4.2-7.5 min.
[0017] The present application provides a kind of automatic cleaning type flow cytometer sample inlet cleaning method, comprising the following steps: automatic cleaning type flow cytometer is sequentially pre-cleaned, formally cleaned and re-cleaned;The cleaning fluid used in pre-cleaning and re-cleaning is Decon 90 cleaning agent;The cleaning fluid used in formal cleaning is sodium hypochlorite solution.
[0018] Compared with the prior art, the present application has the following beneficial effects:
[0019] This invention aims to establish a novel sample inlet cleaning method to remove residues as much as possible, providing a relatively "cleaner" detection microenvironment for sample analysis. In this invention, the cleaning solution used for both pre-cleaning and re-cleaning is Decon 90 cleaning agent. The 90 cleaning solution is a water-based emulsion composed of high-quality anionic and nonionic surfactants, stabilizers, non-phosphate detergents, and alkaline trapping reagents. It is an alkaline concentrate (pH 13+) and does not contain enzymes, EDTA / NTA, or chlorine bleach. It is effective for removing stubborn, adherent substances. The formal cleaning solution used is a sodium hypochlorite solution, which has strong oxidizing properties and can oxidize and destroy organic molecules. Pre-cleaning essentially removes suspended particles adhering to the sample inlet, activating the biofilm at the inlet. Formal cleaning effectively removes deposits from the inner wall of the inlet, and re-cleaning thoroughly washes away broken, suspended biofilm fragments, minimizing background particle count and fluorescence values. This novel cleaning method allows the cell analyzer to operate at its optimal condition. Data from the examples show that after cleaning using this method, the cleanliness of the instrument's sample inlet is significantly improved, and both the quantity and intensity of stray signals are significantly reduced. Attached Figure Description
[0020] Figure 1 A represents the total number of particles detected per 100 μL of cleaning solution in the three stages of pre-cleaning, formal cleaning, and re-cleaning; B represents the change in the median fluorescence intensity (MFI) of the four channels FITC, PE, APC, and PerCP in the three cleaning stages; C represents the box plot of the total number of particles detected per 100 μL of cleaning solution in the three stages of pre-cleaning, formal cleaning, and re-cleaning; D represents the box plot of the median fluorescence intensity (MFI) of the four channels FITC, PE, APC, and PerCP in the three cleaning stages.
[0021] Figure 2 The figure shows the change of SSC-H intensity distribution over time during the pre-cleaning, formal cleaning and re-cleaning stages. The numbers in each figure are the cleaning sequence numbers.
[0022] Figure 3 The graph shows the changes in fluorescence intensity and negative / positive clustering of signals collected at different times during the pre-cleaning, formal cleaning, and re-cleaning stages of the FITC channel. The dashed boxes indicate the gate positions of the positive clusters, and the numbers in each graph represent the number of cleaning cycles.
[0023] Figure 4 This study shows the change in the proportion of positive signals collected during the pre-cleaning, formal cleaning, and re-cleaning stages of the four-fluorescence channel over time. Detailed Implementation
[0024] This invention provides an automated cleaning method for the sample inlet end of a flow cytometer, comprising the following steps:
[0025] The automated cleaning type flow cytometer was subjected to pre-cleaning, formal cleaning, and re-cleaning in sequence; the cleaning solution used for pre-cleaning and re-cleaning was Decon 90 cleaning agent; the cleaning solution used for formal cleaning was sodium hypochlorite solution.
[0026] Unless otherwise specified, all raw materials used in this invention are commercially available products in the field.
[0027] This invention does not impose any special limitations on the specific parameters of the automatic cleaning process; the automatic cleaning can be performed promptly according to the manufacturer's instructions.
[0028] In this invention, the volume fraction of the Decon 90 cleaning agent is preferably 5-10%.
[0029] In this invention, the pre-cleaning is preferably performed 5 times. The purpose of the pre-cleaning is to remove suspended particles and attach them to the injection end, thereby activating the biofilm at the injection end.
[0030] In this invention, the re-washing is preferably performed 5 times. The purpose of the re-washing is to ensure that the broken and suspended biofilm fragments are thoroughly washed away and that the background particle count and fluorescence value are minimized.
[0031] In this invention, the mass fraction of the sodium hypochlorite solution, calculated as active chlorine, is preferably 0.5-1.0%.
[0032] In this invention, the number of formal cleaning cycles is preferably 15, which can effectively remove deposits from the inner wall of the injection end.
[0033] In this invention, the sodium hypochlorite solution is preferably filtered through a 0.22 μm filter membrane before use.
[0034] In this invention, the sample injection for each of the pre-cleaning, formal cleaning and re-cleaning is preferably 100 μL, the sample flow rate is preferably 66~120 μL / min, and the sample flow diameter is preferably 16.8~22.7 μm.
[0035] In this invention, the pre-cleaning time is preferably 4.2 to 7.5 minutes, the formal cleaning time is preferably 12.5 to 22.5 minutes, and the re-cleaning time is preferably 4.2 to 7.5 minutes.
[0036] In this invention, data acquisition is preferably performed simultaneously with cleaning. Preferably, all two scattered light channels (including forward scattering FCS and side scattering SSC) and four fluorescence channels (including FITC, PE, PerCP, and APC) are turned on, with gains set to 500, 420, 493, and 493, respectively. All use the height parameter (H), and the threshold is set to FSC-H greater than 100,000 (the threshold recommended by the manufacturer for cell lines smaller than 20 micrometers or particles of equivalent size). At the same time, the time parameter is used to monitor the change of sample acquisition over time.
[0037] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.
[0038] Example
[0039] 1. Materials and Methods
[0040] 1.1 Reagents
[0041] Decon 90 cleaning fluid: manufactured by Decon Laboratories, UK.
[0042] Sodium hypochlorite solution (Antefomin): Produced by China National Pharmaceutical Group Chemical Reagent Co., Ltd. (52 Ningbo Road, Shanghai). Chemically pure grade, active chlorine (as Cl) ≥ 5.2%, filtered through a 0.22μm filter membrane before use.
[0043] Ultrapure water: purified using an Arium® Comfort Ⅱ, Sartorius (Germany) ultrapure water system, with a resistivity of 18.2 MΩ·cm and a total organic carbon value of less than 5 ppb.
[0044] 1.2 Instruments and Equipment
[0045] The Agilent Novocyte 1040 flow cytometer (formerly ACEA brand), purchased from Agilent Technologies (China) Co., Ltd. (formerly Aisen Biotechnology (Hangzhou) Co., Ltd.), is configured with 2 laser and 4 fluorescence channels: 488nm laser, FITC channel (bandpass filter 530 / 30), PE channel (bandpass filter 572 / 28), PerCP channel (bandpass filter 675 / 30); and 640nm laser, APC channel (bandpass filter 675 / 30). Flow cytometry data analysis software: NovoExpress software, version 1.6.1, Agilent Technologies, Inc. On the day of the experiment, standard microspheres were used for instrument quality control; the instrument status showed all channels were in pass state.
[0046] 1.3 Cleaning Method
[0047] This cleaning method mainly consists of three stages: pre-cleaning, formal cleaning, and re-cleaning. The cleaning solution used for pre-cleaning and re-cleaning is 5% (v / v) Decon 90 cleaning agent; the cleaning solution used for formal cleaning is 0.5% (w / w) sodium hypochlorite solution (calculated as active chlorine).
[0048] Throughout the process, there were 5 pre-washes, 15 formal washes, and 5 re-washes, totaling 25 washes. Each wash consisted of 100 μL of sample. The washing solution was aspirated by the instrument's injection needle via a programmed control, and a high injection rate was used (sample flow rate: 66 μL / min, sample flow diameter: 16.8 μm). The total pre-wash time was 4.2 min, the formal wash time was 12.5 min, and the re-wash time was 4.2 min, for a total of 20.9 min for the entire process.
[0049] Data acquisition was performed simultaneously with the cleaning process. All two scattered light channels (forward scattering FCS and side scattering SSC) and four fluorescence channels (FITC, PE, PerCP, and APC) were opened, with gains set to 500, 420, 493, and 493, respectively. Height (H) values were used for all channel parameters, and the threshold was set to FSC-H greater than 100,000 (the manufacturer's recommended threshold for cell lines smaller than 20 μm or particles of equivalent size). The time parameter was also monitored to track changes in sample acquisition over time. Two-dimensional flow cytometry parameter plots were displayed using contour plots (contour levels of 10%, showing the trend of two-dimensional scatter point distribution density changes).
[0050] 1.4 Data Analysis
[0051] Box plots, positive skewness analysis, interquartile ranges and skewness coefficients, and other statistical data were all obtained using OriginPro 8 software.
[0052] 2 Results and Discussion
[0053] 2.1 Changes in the total number of particles during the three-stage cleaning process
[0054] Figure 1 Figure A shows the trend of the total particle count detected by flow cytometry after each injection of 100 μL of washing solution, as the washing process progresses. The total particle count shows a slight increase in the final stage of the pre-washing phase. This stage primarily utilizes the surfactant, the main active ingredient in the washing agent, to loosen and suspend the adsorbed particles (mainly in biofilm form) on the inner surface of the injection end, thus activating them and laying a foundation for the subsequent formal washing. Figure 1 As shown in Figure A, during the formal cleaning stage, the total particle count initially experienced a significant increase, reaching a peak of 11658 / 100μL, before declining. It plateaued around the seventh cleaning cycle and then continued to decrease with fluctuations. The cleaning solution used in the formal cleaning was a 0.5% sodium hypochlorite solution (calculated as active chlorine), which has strong oxidizing properties. It can directly oxidize and destroy various chemical bonds in the biofilm growing inside the injection end, exerting a more thorough destructive effect. Therefore, the total particle count peaked during the formal cleaning stage. Finally, in the re-cleaning stage, except for a significant increase in the particle count during the first re-cleaning, the count decreased significantly thereafter, by nearly tenfold. This indicates that the re-cleaning solution (5% Decon 90 cleaning agent) almost completely washed away the debris generated by the oxidation of the injection end by the sodium hypochlorite solution.
[0055] Figure 1 Figure C shows the median, upper and lower quartiles, mean, and outliers of the total particle count for three washing stages. The distribution of the total particle count in all three stages exhibits a positively skewed distribution; therefore, under the actual experimental conditions, the median better reflects the actual situation. The median total particle counts for the three stages are 139, 3733, and 295, respectively. Converted to volume, this results in an equivalent particle concentration of 2.55 × 10⁻⁶. 3 The concentration was 10c / mL, and the signal was almost entirely in the debris and noise region, while the reasonable concentration range for preparing flow cytometry samples is 10c. 6 ~10 7 The number of particles per mL is negligible for routine flow cytometry experiments. Most flow cytometry experiments, especially those involving tiny particles such as bacteria and cyanobacteria, rely on analyzing the varying intensity of fluorescent labeling on the particles to arrive at conclusions. Therefore, we subsequently analyzed the changes in signal intensity across the four fluorescence channels of our flow cytometer during the washing process.
[0056] 2.2 Changes in the intensity of the four-channel fluorescence signal during the three-stage cleaning process
[0057] This flow cytometer is widely used in toxicology, cell biology, immunology, and environmental microbiology. Common experiments included are not limited to cell line apoptosis and necrosis, cell cycle, mitochondrial membrane potential, peripheral blood mononuclear cell typing, helper T cell differentiation, bacterial viability and death, and cyanobacterial growth. The fluorescence channels involved include all configured channels: FITC, PE, PerCP, and APC. Therefore, the changes in the signals of each fluorescence channel during the washing process are worth noting. Figure 1 Figure B shows the changes in signal intensity of the particles in the four fluorescence channels as the washing process progresses, with the vertical axis MFI representing the median fluorescence intensity.
[0058] from Figure 1 As shown in Figure B, the upward trend of the median fluorescence intensity (MFI) of the four fluorescence channels during the pre-cleaning stage further illustrates the activating effect of pre-cleaning on the residue at the injection end. Entering the formal cleaning stage, the significant decrease in MFI across all four channels during the first cleaning process can be explained by the strong bleaching properties of the sodium hypochlorite solution, which immediately bleached and defluorinated a small number of suspended fluorescent particles. Subsequently, as the biofilm released a large amount of fragmented particles, the MFI reached its peak in the fifth formal cleaning, then fluctuated steadily at a low level, finally dropping to its lowest level during the re-cleaning stage. After cleaning, the MFI values of all four fluorescence channels gradually decreased, falling below those of the pre-cleaning stage, indicating that the interference of the residue at the injection end on the fluorescence channels was significantly reduced after treatment with this cleaning method.
[0059] Box plot statistics of MFI for four fluorescence channels were examined during the pre-cleaning, formal cleaning, and re-cleaning stages. Except for FITC and ACP channels, which showed a near-normal distribution during the re-cleaning stage, the MFI distributions for all other channels were positively skewed across all three stages. Figure 1 As shown in the middle D, the median MFI of the four fluorescence channels was pre-cleaning > formal cleaning > re-cleaning, indicating that all three cleaning stages played a significant role. Moreover, the interquartile range values of the four fluorescence channels dropped significantly to the lowest level during the re-cleaning stage, indicating that the MFI of each fluorescence channel was not only low during the re-cleaning stage, but also exhibited high stability.
[0060] This flow cytometer has a wide range of applications. When determining the concentration of bacteria and other microorganisms, it extensively uses fluorescent probes such as SYBR Green and SYTO9 as nucleic acid indicators, all of which use FITC fluorescence channels. Additionally, many experiments involving apoptosis and necrosis, bacterial mortality, and cell cycle analysis utilize PI dyes, which are detected in both PE and PerCP channels. Furthermore, algal detection can leave residues at the injection end, and algal chlorophyll exhibits significant autofluorescence in the APC channel. Therefore, all of these fluorescence channels are affected by residues at the injection end. However, after cleaning using this method, interference is almost completely eliminated.
[0061] 2.3 Changes in fragment complexity with washing time
[0062] In flow cytometry experiments, the most fundamental parameter is the scattered light signal parameter, which is divided into forward scattering (FSC) and side scattering (SSC). The angles of the scattered light signals collected by the detectors of the two are different (0º and 90º). They play an extremely important role in the application of flow cytometry and are generally used for the initial selection of cells.
[0063] In this study, forward scattering (FSC) represents the relative size of the particles, while side scattering (SSC) represents the complexity of the particles, i.e., the complexity of their surface or internal structure. Larger internal particle sizes or greater heterogeneity result in a stronger SSC signal. This invention monitors the scattering signal simultaneously with the sample inlet cleaning process. The overall FSC-H variation was minimal (data not shown), indicating that the size of the eluted fragments and clumps did not change significantly. However, the SSC-H intensity distribution exhibited interesting changes across the three cleaning stages. For example... Figure 2 As shown, during the pre-cleaning stage, SSC-H showed a clear upward trend with increasing cleaning time (number of cleaning cycles). Upon entering the formal cleaning stage, SSC-H reached its peak and then fluctuated steadily at a low level. Figure 2 (Formal cleaning). Entering the re-cleaning stage ( Figure 2 During the secondary cleaning process, SSC-H showed a decreasing trend. These results demonstrate the importance of secondary cleaning; while the oxidative damage effect of the primary cleaning plays a decisive role, the removal of residual adsorbed debris still requires the participation of surfactants.
[0064] 2.4 Changes in positive groups of fluorescent channels with washing time
[0065] In flow cytometry analysis, an important aspect is the change in the proportion of positive clusters in the fluorescence channels. Therefore, we examined the changes in the proportion of positive clusters in each channel as washing progressed, and used the FITC channel as an example for illustration. Figure 3This paper demonstrates the changes in signal strength and positive particle distribution in the FITC channel over three cleaning stages as cleaning time progresses. During the pre-cleaning stage, the proportion of positive particles in the FITC channel shows an increasing trend with the number of cleaning cycles. Quantitative statistics of the positive particle proportion are presented below. Figure 4 It can be seen that all four channels showed a significant increase during the pre-cleaning stage, which fully demonstrates the loosening and activation effect of pre-cleaning on the adsorbate at the injection end.
[0066] Entering the formal cleaning stage, by Figure 3 It is evident that a clear trend of continuous positive results emerged starting from the fourth wash, with distinct clusters of positive results appearing in the fifth wash. Figure 4 It can be seen that the proportion of positive groups in each channel dropped to a stable minimum level after reaching the maximum peak.
[0067] During the re-washing phase, the proportion of FITC-positive clusters showed a steady downward trend. Figure 4 As can be seen, the proportion of positive groups in all four channels eventually dropped below 10%. This demonstrates that the three-stage cleaning process effectively reduced the proportion of positive groups in each fluorescence channel.
[0068] As shown in Table 1, the differences in the total number of particles and the proportion of positive groups in the four fluorescence channels (FITC, PE, APC, and PerCP) after the instrument's automatic cleaning and the cleaning method of this invention were compared. It was found that all five parameters decreased after cleaning by this method compared to after automatic cleaning, indicating that this method does indeed play a further cleaning role.
[0069] Table 1. Test results of total particle count after automatic cleaning by the instrument and cleaning by the method of this invention.
[0070] Total particle count ( / 100 μL) Proportion of FITC-positive population (%) Proportion of PE-positive population (%) Proportion of APC-positive population (%) Proportion of PerCP-positive population (%) After automatic washing 866 11.78 15.47 4.39 9.93 After washing by the method of the present application 230 9.57 10.87 4.35 7.39
[0071] In summary, the detection of pathogenic microorganisms in environmental samples is receiving increasing attention, including but not limited to viruses and bacteria. Flow cytometry, as a high-throughput, rapid, and easy-to-operate method, is gaining popularity among researchers in related fields. In recent years, the demand for using flow cytometry to analyze trace microorganisms in environmental water samples has been growing, with concentrations often reaching 10⁻⁶. 3 The concentration of samples per mL or even lower places higher demands on the cleanliness of the instrument itself. This invention provides a new cleaning method. Experimental results show that the method significantly removes residues at the sample inlet. For quantitative detection of low-concentration samples, it can minimize non-specific interference at the sample inlet, providing a cleaner microenvironment for sample detection and making the results more reliable. This method can serve as a reference for flow cytometer managers and operators.
[0072] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A method for cleaning the sample inlet end of an automated flow cytometer, characterized in that, Includes the following steps: The automated cleaning type flow cytometer was subjected to pre-cleaning, formal cleaning, and re-cleaning in sequence; the cleaning solution used for pre-cleaning and re-cleaning was Decon 90 cleaning agent; the cleaning solution used for formal cleaning was sodium hypochlorite solution. The volume fraction of the Decon 90 cleaning agent is independently 5-10%; The pre-cleaning is performed 5 times; The sodium hypochlorite solution has a mass fraction of 0.5% to 1.0% based on active chlorine. The formal cleaning process is repeated 15 times. Decon 90 cleaning agent is a water-based emulsion composed of anionic and nonionic surfactants, stabilizers, non-phosphate detergents, and alkaline trapping agents. It is an alkaline concentrate with a pH of 13+ and does not contain enzymes, ethylenediamine acetate / hypochlorous acid triacetic acid, or chlorine bleach. It is effective for removing stubborn, adherent substances. The cleaning solution used in the formal cleaning is a sodium hypochlorite solution, which has strong oxidizing properties and can oxidize and destroy organic molecules. Pre-cleaning essentially removes suspended particles that adhere to the sample inlet, activating the biofilm at the inlet. Formal cleaning effectively removes deposits from the inner wall of the inlet. Re-cleaning thoroughly washes away broken, suspended biofilm fragments, minimizing background particle count and fluorescence value.
2. The automatic cleaning method for the sample inlet end of a flow cytometer according to claim 1, characterized in that, The re-cleaning process is performed 5 times.
3. The automatic cleaning method for the sample inlet end of a flow cytometer according to claim 1, characterized in that, The sodium hypochlorite solution was filtered through a 0.22 μm filter membrane before use.
4. The automatic cleaning method for the sample inlet end of a flow cytometer according to claim 1, characterized in that, Each injection for the pre-cleaning, formal cleaning, and re-cleaning processes is 100 μL, with a sample flow rate of 66–120 μL / min and a sample flow diameter of 16.8–22.7 μm.
5. The automatic cleaning method for the sample inlet end of a flow cytometer according to claim 1 or 4, characterized in that, The pre-cleaning time is 4.2~7.5 min, the formal cleaning time is 12.5~22.5 min, and the re-cleaning time is 4.2~7.5 min.