流式细胞仪 z-bo 2020-08-14 534

　　Commissioning and calibration

　　The flow cytometer must be carefully adjusted before use, and even during use, to ensure the reliability and optimality of the work. The debugging items are mainly laser intensity, liquid flow velocity and optical path in the measurement area.

　　(1) Laser intensity adjustment: In addition to adjusting the angle of the reflector to adjust the laser light output of the required wavelength, it is also necessary to combine the spectral curve on the display to maximize the laser intensity output.

　　(2) Liquid flow speed adjustment: The gas pressure can be adjusted to obtain a stable liquid flow speed through the digital display supervision of the console.

　　(3) Optical path adjustment in the measurement area: This is the key to debugging. It is necessary to ensure that the liquid flow, laser beam, and 90-scattering measurement photoelectric system in the measurement area are perpendicular to each other, and the intersection point is small. Generally, it can be completed in calibration with standard fluorescent microspheres.

　　The quantity measured in flow cytometry is a relative value, so the system needs to be calibrated or calibrated before or during use, so that the absolute meaning can be obtained through relative measurement. Therefore, the calibration in FCM has dual functions: the collimation adjustment of the instrument and the quantitative scale. The standard sample should be stable, the shape of the formed component should be a relatively uniform spherical shape, the sample has good dispersion performance, and is economical and easy to obtain. Standard fluorescent microspheres are commonly used as non-biological standard samples, and chicken red blood cells are used as biological standard samples.

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　　The microspheres are made of resin material, or labeled with fluorescein, or not labeled with fluorescein. The preparation process of the chicken red blood cell standard sample used is as follows: take 3.8% citric acid or heparin anticoagulated chicken blood (anticoagulant: chicken blood=1:4), wash 3 times with PBS, and then use 5-10ml 1.0% glutaraldehyde was mixed with the cleaned chicken red blood cells, aldehyde-formed by shaking at room temperature for 24 hours, and finally washed with PBS, and stored in a refrigerator at 4°C for later use. It should be pointed out that because there is no fluorescent staining, the measured light signal is the autofluorescence of chicken hemoglobin.

　　1.4.2. Operation and use of the instrument

　　(1) Turn on the power and warm up the system;

　　(2) Open the gas threshold, adjust the pressure to obtain a suitable liquid flow speed; open the light source cooling system;

　　(3) Add deionized water to the sample tube to flush the nozzle system of the liquid flow;

　　(4) Using calibration standard samples, adjust the instrument so that based on the adjustment of laser power, photomultiplier tube voltage, and amplifier circuit gain, the fluorescence intensity scattered by 0 and 90 is the strongest, and the coefficient of variation is required to be the smallest;

　　(5) Select flow rate, number of cells to be measured, measurement parameters, etc., and measure samples and control samples under the same working conditions; at the same time, select the display mode of data on the computer screen, so that the measurement process can be intuitively grasped;

　　(6) After the sample is measured, rinse the liquid flow system with deionized water;

　　(7) Because the experimental data has been stored in the computer hard disk (some machines are also equipped with an optical disk system, which has a larger storage capacity), the gas and measuring devices can be turned off, and the computer can be used for data processing alone;

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　　(8) Print out the desired result.

　　1.4.3 Precautions in operation and use

　　(1) The photomultiplier tube requires stable working conditions. After exposure to strong light, it needs a longer period of "dark adaptation" to eliminate or reduce part of the dark current background before it can work; in addition, pay attention to magnetic shielding;

　　(2) The light source must not be turned off and on again within a short period of time (usually about 1h); the light source must be preheated and the cooling system must be working properly;

　　(3) The fluid flow system must keep the fluid flowing at all times to avoid bubble embolism. The sheath fluid used must be filtered and disinfected before use; pay attention to selecting the appropriate filter system and amplifier type according to the change of the measurement object;

　　(4) The special intensity requires a control group for each measurement