If cells are a grand parade, flow cytometry is the “super intelligent camera” that can simultaneously record everyone’s height, clothing color, and even facial expressions.
The principle of flow cytometry
Flow cytometry is a fluorescence-based detection method that simultaneously measures multiple indicators from individual cells, such as cell population counting and protein abundance. It can simultaneously analyze the physical characteristics (such as size) and chemical characteristics (such as surface and intracellular specific protein expression) of cells as they pass through the instrument, enabling precise analysis of complex cell populations.
Principle: A technique that utilizes laser excitation of high-speed, uniformly moving particles with various fluorescent markers, and detects the resulting scattered light signals and fluorescence signals to reflect various parameters of the particles. Specific traits of the particles are sorted out for further analysis or cultivation.
Figure 1 Flow Cytometer
Characteristics of flow cytometry
- research subject
Population of cells (proportion, average fluorescence intensity, etc.)。
- Fast analysis speed and high sorting accuracy
achievable1*105Cells/second, with a purity of over 99%。
- Capable of simultaneously detecting multiple types of information from cells
It can simultaneously detect information including but not limited to the cell membrane, nucleus, cytoplasm, and other relevant details.
The detection process is as follows:：
Precautions：
- In flow cytometry experiments, it is usually necessary to collect a sufficient number of cells with complete viability as samples for detection. For different types of samples, there are appropriate methods to process them into single-cell suspensions suitable for the experiment;
- After sample processing, cell counting is required to ensure that the cell quality is suitable for the experiment;
- The purpose of vital staining is to eliminate interference from dead cells;
- The function of Fc blockers is to block specific Fc receptors present on the surface of macrophages and other cells, thereby reducing specific staining;
- For experiments that require simultaneous detection of cell surface and intracellular antigens, stain the cell surface antigens before fixation/membrane permeabilization. If surface antigen staining is performed after cell fixation, appropriate antibodies need to be determined through experimentation;
- Fluorescein is prone to quenching under strong light exposure, hence the preservation and incubation of antibodies should be conducted in the dark;
- If the sample cannot be loaded onto the instrument on the same day it is prepared, it can be fixed with 4% paraformaldehyde and temporarily stored in the loading buffer at 4℃ in the dark;
- Adjust the voltage of each channel of the instrument based on the signal from the negative control to ensure that all sample signals can be fully displayed.
Practical application of flow cytometry
The KCI•KMQ Molecular Biology Platform boasts advanced technical means and extensive project experience. It is capable of handling biochemical detection indicators with a daily detection volume exceeding 300, and also offers a variety of molecular biology detection methods, including flow cytometry analysis, qPCR, ELISA, WB, IP/Co-IP, and more. Currently, the company has established extensive long-term collaborations with numerous renowned pharmaceutical companies and scientific research institutions both domestically and internationally, laying a solid foundation for the development of innovative drugs.
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About KCI Biotech
KCI Biotech and its wholly-owned subsidiary, KMQ Biotech and KAL Biotech are CROs specializing in providing preclinical pharmacodynamics and durability evaluation services for pharmaceutical research and development. The company owns AAALAC-accredited state-of-the-art laboratory animal facilities in Suzhou and Nantong,with a total area of more than 40,000m²,which can meet the needs of various types of preclinical research. The company has a full range of laboratory animal platforms including rats, mice, hamsters, guinea pigs, rabbits, cats, ferrets, dogs, pigs, sheeps and non-human primates. The in vivo pharmacolofical department supplies more than 400 animal disease models for human medicine development research. The BSL2/ABSL2 department supplies more than 100 pathogenic microorganisms, including bacteria, viruses, and fungi for human medicine development research, veterinary medicine and pet medicine. The company also supplies pathology, toxicology, pharmacokinetics, cellular and molecular biology, and medical imaging services. The company has already cooperated with more than 1,000 famous domestic and international pharmaceutical companies , and completed more than 2000 domestic and international projects .
With a professional R&D team, rich experience in in vivo pharmacology and pharmacodynamics evaluation, and a professional spirit of integrity and excellence, KCI/KMQ/KAL will provide a full range of preclinical drug discovery and development services for global pharmaceutical companies and research institutions, and establish a professional and efficient drug discovery and evaluation system.
