Flow-cytometric Analysis

2024-01-03 Hits(210)

FACS is a selective technique for the purification of known phenotypic cell populations. FACS is the preferred method when a desired population of very high purity is required, when the target cell population expresses very low levels of identification markers, or when the cell population needs to be separated based on differential marker density. In addition, FACS is the only purification technique available to isolate cells based on internal staining or intracellular protein expression, such as gene-modified fluorescent protein labeling. FACS allows purification of individual cells based on size, particle size, and fluorescence.


1.How Flow Cytometry Works


Flow cytometry works by allowing cells to be tested into a single cell suspension, which requires specific fluorescent staining. The scattered light and excited fluorescence generated by the laser beam were collected under irradiation for analysis. The intensity of these fluorescent signals represents the level of expression of antigens on the cell surface or the concentration of substances in the cell nucleus, which can be converted into an electrical signal received by a photomultiplier tube and then converted into a digital signal that can be recognized by a computer via an analog/digital converter. Cell ordering is achieved by separating droplets containing single cells. The nozzle flow chamber is equipped with UHF transistors. After charging, it vibrates and breaks up the ejected liquid flow into uniform droplets. The cells to be measured are dispersed in these droplets, which are either positively or negatively charged, and separated in an electric field. In contrast to MACS, FACS can not only analyze cell morphological characteristics, such as size and cell particle size detection through forward and lateral scattering, but also detect multiple surface markers at the same time, allowing better localization of target cells for further sorting.


2.Flow Cytometry Procedure


(1) Prepare a single-cell suspension of the initial cell population;

(2) Enrichment of desired cell populations by mass purification methods, such as complement depletion or magnetic sorting. The main advantage of the concentration step is that it reduces the sorting time;

(3) Wash the cells once with dye buffer;

(4) Discard the supernatant and resuspend the cells in a dye buffer with a concentration of up to 50x106 for effective staining;

(5) For cells expressing high levels of FcR, block the receptor using appropriate blocking methods. One option is to use monoclonal antibodies that bind to FcγR on ice for 10-15 minutes;

(6) Add appropriate mAb (predetermined concentration) to stain the desired cell population, incubate in the dark on ice for 20-30 minutes, and then wash twice with the dye buffer;

(7) If a directly conjugated antibody is not used, repeat step 6 with an appropriate second antibody or streptavidin conjugate;

(8) After washing, the cells were re-suspended in the medium, and the cell concentration was determined with an important dye such as Trypan blue;

(9) Adjust the cell concentration to 15-20x106/ml. For clusters of cells that may clog the instrument during sorting, filter the cells through a strainer;

(10) Set up and optimize the cell sorting machine;

(11) Use a negative control sample and a single positive control to compensate. In order to eliminate spectral overlap between the two detectors, compensation is required. It is important to note that compensation is not foolproof and can be adversely affected by the way certain markers are stained, as well as by cell fluorescence with low autofluorescence. Low autofluorescence results in poor resolution between dim and negative populations;

(12) Record experimental samples to be classified, and use gating tools and subset setting methods to define groups of interest;

(13) Once the gate is identified, the gate can be selected and sorted into the external collection tube. Depending on available instruments, up to four phyla (or populations) can be sorted at a time;

(14) Run the experimental sample tube at 4℃, open the deflector plate, and classify the sample. 5x105-1.5x106 cells can be sorted into 12x75mm test tubes, and 1.5x106-4.5x106 cells can be sorted into 15mL cone test tubes.

(15) After obtaining the required number of cells, manually stop sorting;

(16) A post-sorting analysis was performed to determine the purity of the sorted cell population.


3. Fluorescent Signal Source


For the desired cell population analyzed using flow cytometry and separated by sorting, a light signal is required. There are two types of light, scattered light that is parallel or orthogonal to the path of the excited beam, and light that is emitted as fluorescence. Like all cells, plant protoplasts contain endogenous compounds that fluoresce themselves when excited, especially at shorter laser wavelengths. Protoplasts from green tissues (aerial organs) exhibit high levels of chlorophyll autofluorescence due to excitation of photopigments in leaf green.

Another approach to fluorescent labeling is to stain with an exogenous fluorescent dye, either a directly reactive substance, a fluorescent compound that can be absorbed by plants as a fluorescent hormone analogue, or a fluorescent compound attached to an antibody ligand, the latter being widely used in flow cytometry of mammalian cells.


4.Flow Separation Technique is used for Screening Nucleic Acid Aptamers


FACS is a sensitive, high-throughput, automated method that is widely used to separate cells from other subpopulations of cells, distinguishing between living and dead cells based on different light-scattering properties or their ability to convert dyes. Therefore, it can eliminate the effects of non-specific adsorption of dead cells. In addition, the screening process can be monitored online through FACS, and the severity of online screening can be well controlled by setting the sorting gate. Facs-assisted SELEX assays can be divided into two categories. The first approach is to bind the aptamer to a large target, such as cells and bacteria, directly using FACS to isolate and collect the large target of bound nucleic acids. The second is the aptamer particle SELEX (APs-SELEX), which allows each aptamer to form an aptamer particle that contains many copies of a single aptamer on its surface. This method has the advantages of high efficiency and high speed. In addition, it can eliminate unwanted enrichment of non-specific aptamers. However, the preparation of aptamer particles is very complex, and a round of routine selection using magnetic beads is required to reduce the number of libraries. An innovative AgFACS-SELEX (AGNP-assisted FACS SELEX) method has been developed to eliminate preenrichment and reduce the number of selections. This method has the following advantages: Firstly, it is found that AgNP can enhance the fluorescence intensity of Cy5, and AgNPCy5 can hybridize with the library to select non-self-hybridizing libraries, which significantly improves the screening efficiency and reduces the selection times. The mechanism of fluorescence enhancement can be found in our previous report. Second, each round of selection can be monitored online by FACS. By setting the sorting gate, you can recycle and enrich the high affinity of the selected library. Finally, the method selection does not require pre-enrichment of libraries and preparation of aptamer particles. In addition, nanoparticle based screening signal amplification has not been reported by FACS-SELEX.


KMD Bioscience is able to supply its customers with suspension cells (e.g., B cells), adherent cells or single-cell suspensions isolated from solid tissues, blood, various body fluids and other biological particles (e.g. Intracellular cytokine) flow cytometry services can also provide single B cell isolation and sorting for single B cell screening techniques, and can analyze a variety of parameters such as intracellular DNA, RNA, proteins and cell surface antigens.