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Streaming detection

Streaming detection

First, project introduction
Flow cytometry is the use of flow cytometry to perform spectral identification, cell sorting, and counting of cells or particles. It is widely used to analyze the expression levels of cell surface and intracellular molecules, identify different cell types of cell populations in samples, and evaluate various The purity of cell subsets and analysis of cell size and contained particles. Mainly use fluorescently labeled antibodies to detect protein levels, or fluorescent dyes that bind specific cell molecules, such as the combination of PI and DNA.

Second, application
Commonly used in the identification and level analysis of cell surface molecules such as surface markers to identify, qualitatively or quantify specific groups of cells, can detect protein expression levels in cells, can use specific dyes to analyze cell cycle and apoptosis, and use mitochondrial potential dyes Analyze the mitochondrial membrane potential and use dyes such as DHE to detect the level of intracellular ROS.

Third, the flow cytometer channel
Beckman Coulter CytoFLEX flow cytometer; optical parameters: FSC (forward scatter), SSC (side scatter), 4-color fluorescence channel; data acquisition software: CytExpert.

Excitation wavelength Aisle Emission wavelength Common dyes
488 nm FL1 525±20 nm FITC、EGFP、CFSE、AlexaFluor 488
FL2 585±21 nm PE、PI、DsRed
FL3 690±25 nm PerCP-Cy5.5、PC5
638 nm 660±10 nm APC、Alexa Fluor 647

Fourth, the results show

cell cycle


ROS detection

JC-1 Mitochondrial membrane potential

Fifth, sample storage and transportation

1. Precautions for receiving streaming samples

① Apoptosis

requires live cells (no samples from other places). If the cells are fluorescent, be sure to make a note on the transfer list and culture plate, otherwise treat as non-fluorescence. If the test results are unavailable and samples are wasted as a result, we will not be responsible and will not accept refunds.

②Cell cycle

The    cycle requires a large amount of cells, and the 6-well plate must have a confluence of more than 70%, but not more than 90%. Samples from the field need to be fixed overnight with pre-cooled 70% ethanol (adherent cells need to be digested and fixed), centrifuge to discard the fixative, add 1ml of PBS to resuspend, seal with a plastic film, and ship to the laboratory at 4°C.

③Flow JC-1

requires live cells (no samples from other places). The cell confluence is higher than 70% (6-well plate), and 2 more blank holes are provided for negative and positive controls. If the cell has red or green light, it cannot be detected.

④ Streaming ROS

requires live cells (no samples from other places). The cell confluence is higher than 70% (6-well plate), and 2 more blank holes are provided for negative and positive controls. If the cell is green, it cannot be detected.

⑤Cell surface resistance

Determine in advance whether the company has the corresponding flow antibody (histochemical antibody is not available), other antibodies need to be provided by the customer (no samples from other places).

⑥ Streaming triple standard

Inform the specific number and type of samples (blood or spleen~) in advance. Blood samples are collected in anticoagulant tubes (at least 1.5 ml for intracellular staining and about 1 ml for epitope). Take care to prevent coagulation and store and transport at 4°C. Blood samples can only be stored at 4°C for one week at most. The longer the time, the worse the results will be. After the blood is taken, it must be sent to the laboratory as soon as possible; when taking the spleen, try to bring the animal to the laboratory, and start the experiment directly after taking the material from the laboratory. If the animal cannot be taken to the laboratory, take the material (the entire spleen) and place the tissue in Store in an EP tube or centrifuge tube containing PBS at 4°C. Cell extraction must be completed within 6 hours after sampling. Note that some test indicators (Treg, TH1, TH2, etc.) need to be extracted aseptically. Samples from other places will not be accepted.

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