Method for detecting white blood cells by flow cytometry

Abstract

The invention relates to a novel method for detecting white blood cells through a flow cytometry and application of the novel method in the medical field. The innovation of the method is that high-throughput, high-precision and multi-parameter analysis is realized by optimizing sample treatment, cell marking, instrument setting and data processing, and more reliable and accurate data is expected to be provided in clinical diagnosis, disease monitoring and treatment strategy formulation.

Description

Technical Field

[0001] The invention belongs to the technical field of cell detection, and in particular relates to a method for detecting leukocytes by flow cytometry. Background Art

[0002] Over the past few decades, flow cytometry has become a core technology in many fields, including immunology, cell biology, oncology, and clinical diagnostics. However, as science continues to advance, the needs of researchers and clinical laboratories for single-cell analysis are increasing. These needs include, but are not limited to:

[0003] Single-cell heterogeneity research: In cancer research and immunology, understanding the heterogeneity between different cell subpopulations is crucial for a deeper understanding of disease mechanisms and the development of therapeutic strategies. Traditional flow cytometry has limitations in this regard, and higher resolution and accuracy are needed.

[0004] Clinical diagnosis: Single-cell analysis has great potential in clinical diagnosis, such as detection of tumor markers, immune phenotype identification, and disease monitoring. However, existing methods are challenging in terms of diagnostic complexity and cost-effectiveness.

[0005] Drug screening: Using single-cell analysis to evaluate the effects of drugs on different cell subpopulations can help personalized medicine and drug development. However, existing analytical methods lack the ability of high throughput and multi-parameter analysis.

[0006] Disease Mechanism Research: Single-cell analysis can be used to study the pathogenesis and pathophysiology of diseases, but more precise and reproducible methods are needed.

[0007] The detailed description of the background technology of the present invention is intended to highlight the challenges currently faced by flow cytometry in these key areas and to address the need to solve these challenges. The present invention provides an improved flow cytometry detection method that better meets the above needs by optimizing various steps to improve resolution, accuracy, throughput and economy.

[0008] Specifically, the innovation of this method includes but is not limited to the following aspects:

[0009] Optimization of sample preparation: Use improved cell fixation and sample handling methods to ensure cell integrity and stability, especially in complex samples.

[0010] High-affinity labels: Use highly specific fluorescent labels or antibodies to ensure specificity and intensity of labeling and reduce background signals.

[0011] Instrument Setup and Calibration: Optimize the flow cytometer’s laser configuration, fluorescence detection channels, and instrument calibration to ensure data accuracy and consistency.

[0012] Advanced Data Processing: Utilize advanced computer software for automated data cleaning, cell identification, parameter determination, and data visualization to obtain interpretable results.

[0013] In summary, the technical background of the present invention has highlighted the limitations and needs of existing flow cytometry methods, providing a basis for the rationality and practical application prospects of the present invention. Through improved methods, the present invention is expected to promote further development in the field of single-cell analysis, provide more solutions for in-depth research and solving complex biological problems, and play a greater role in clinical diagnosis and drug development. Summary of the invention

[0014] The purpose of the present invention is to solve the above technical problems and provide a method for detecting leukocytes by flow cytometry.

[0015] The above technical objectives of the present invention are achieved through the following technical solutions: specifically comprising the following steps:

[0016] S1: Resuspend the cell sample at 1x10^6 cells / mL in cell assay buffer to ensure the appropriate cell number is available for analysis.

[0017] S2: Use high-affinity fluorescent markers or antibodies to mark the target protein or molecule on the cell surface or inside. The choice of marker depends on the purpose of the study. Anti-CD4 antibodies are used to mark T cells, and anti-CD8 antibodies are used to mark another cell subset. The marker concentration is usually 1μg / ml.

[0018] S3: The cell sample is introduced into the flow cytometer, which includes the following settings:

[0019] A 488 nm laser was used to excite the fluorescence of the marker.

[0020] Forward scatter (FSC) and side scatter (SSC) angles are used to measure cell size and complexity.

[0021] The fluorescence intensity of different markers was measured by multi-channel fluorescence detection.

[0022] Each marker is detected using a specific fluorescence channel, for example, the CD4 marker uses the fluorescence 1 channel (FL1-A) and the CD8 marker uses the fluorescence 2 channel (FL2-A).

[0023] S4: Collect cell samples to be analyzed from blood, tissue or cell culture.

[0024] S5: Data processing and analysis: Analyze, compare, and quantify the data obtained by flow cytometry. Computer software or custom algorithms can be used to process the data and extract information about cell surface proteins, such as expression levels, expression patterns, and distribution of cell subpopulations.

[0025] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0026] 1. High sensitivity and high resolution: This method combines flow cytometry and specific labeled antibodies to achieve high-sensitivity and high-resolution identification of cell surface proteins. By using carefully designed and selected labeled antibodies, accurate recognition and labeling of target proteins can be ensured, thereby improving the accuracy of identification.

[0027] 2. Multi-parameter analysis: This method can label multiple proteins at the same time and perform multi-parameter analysis by flow cytometry. By using different fluorescently labeled antibodies, the expression of multiple proteins can be detected and analyzed simultaneously. This provides researchers with more comprehensive information on cell surface proteins and helps reveal details of cell function and subpopulation distribution.

[0028] 3. Rapid and high-throughput: This method is fast and high-throughput, and can quickly process large quantities of cell samples and analyze them. The high-speed data acquisition and processing capabilities of flow cytometers, as well as automated sample processing systems, make large-scale cell surface protein analysis possible. DETAILED DESCRIPTION

[0029] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Embodiment 1:

[0031] A method for detecting leukocytes by flow cytometry, the flow cytometry surface protein identification and analysis method comprising the following steps:

[0032] S1: Collect cell samples to be analyzed from blood, tissue or cell culture. Peripheral blood samples can be collected using anticoagulated blood vessels.

[0033] S2: Centrifuge the blood sample to separate the red blood cells and collect the white blood cell pellet. Suspend the white blood cells in phosphate buffered saline and adjust the cell density to about 1x10^6 cells per ml;

[0034] S3: Use CD3 antibody (to mark T cells) and CD19 antibody (to mark B cells);

[0035] S4: Add the selected antibody or fluorescent marker to the cell sample to allow it to bind specifically to the target cells. The marker concentration is usually 1 μg / ml and incubate at room temperature for 30 minutes;

[0036] S5: Use light scattering to detect forward scatter (FSC) and side scatter (SSC), the excitation light source is a 488nm laser, the detection of fluorescent marker 1CD3 is carried out in the FL1 channel, the detection of fluorescent marker 2CD19 is carried out in the FL2 channel, and the corresponding negative control samples are set to calibrate the instrument;

[0037] S6: Exclude doublets: Use forward scatter width (FSC-W) and forward scatter height (FSC-H) to exclude doublets, ensure that each event is counted only once, calculate the percentage of CD3-positive T cells and CD19-positive B cells in total cells, and identify different cell subpopulations.

[0038] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for detecting leukocytes by flow cytometry, characterized in that: The following steps are involved: S1: Collect cell samples to be analyzed from blood, tissue or cell culture. Peripheral blood samples can be collected using anticoagulated blood vessels. S2: Centrifuge the blood sample to separate the red blood cells and collect the white blood cell pellet. Suspend the white blood cells in phosphate buffered saline and adjust the cell density to about 1x 10^6 cells per ml; S3: Use CD3 antibody (to mark T cells) and CD19 antibody (to mark B cells); S4: Add the selected antibody or fluorescent marker to the cell sample to allow it to bind specifically to the target cells. The marker concentration is usually 1 μg / ml and incubate at room temperature for 30 minutes; S5: Use light scattering to detect forward scatter (FSC) and side scatter (SSC), the excitation light source is a 488nm laser, the detection of fluorescent marker 1CD3 is carried out in the FL1 channel, the detection of fluorescent marker 2CD19 is carried out in the FL2 channel, and the corresponding negative control samples are set to calibrate the instrument; S6: Exclude doublets: Use forward scatter width (FSC-W) and forward scatter height (FSC-H) to exclude doublets, ensure that each event is counted only once, calculate the percentage of CD3-positive T cells and CD19-positive B cells in total cells, and identify different cell subpopulations.

2. The method for detecting leukocytes by flow cytometry according to claim 1, characterized in that: The labeled antibody has specificity and high affinity and is used for specific labeling and staining of cell surface proteins.

3. The method for detecting leukocytes by flow cytometry according to claim 1, characterized in that: The multi-parameter analysis of flow cytometry includes multi-channel excitation and detection to obtain the fluorescence signal of protein markers.

4. The method for detecting leukocytes by flow cytometry according to claim 1, characterized in that Data processing and analysis include parsing, comparison and quantitative analysis of flow cytometry data.