Single cell counting method and system
A single-cell and cell-based technology, applied in the field of metrology, can solve the problems of affecting the accuracy of quantitative results, reducing the repeatability of quantitative results, and uneven cell distribution, so as to achieve the effects of improving detection sensitivity, direct absolute measurement, and accurate quantitative results
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Embodiment 1
[0050] The system used for the flow cytometric single cell counting method is mainly composed of a liquid flow system, a microfluidic chip, a fluorescence detection device and a data processing device, and further includes a data display device. The flow cytometric single-molecule counting platform composed of the above-mentioned system has a schematic diagram of its workflow as shown in figure 2 As shown, a microfluidic chip 7 , an objective lens 8 , a dichroic mirror 9 , and a light source 10 .
[0051] The microfluidic chip device for single cell counting consists of a syringe pump, a liquid storage tank 1, a drainage catheter 2, and a microfluidic chip 7. The microfluidic chip 7 contains a cylindrical sample inflow port 3 and an output port 5, Fluorescence detection area 4, and a flow adjustment device for adjusting the flow rate of the sample flow. The built-in flow channel 11 of the microfluidic chip 7 is connected with the sample inflow port and the output port respec...
Embodiment 2
[0064] The concrete operation of the inventive method is as follows:
[0065] 1. Sample preparation: Use phosphate buffered saline (PBS) filtered through a 0.22 μm filter membrane to dissolve and dilute high-concentration cell samples; add an appropriate amount of fluorescent antibody to the cell sample to fully label the cells; the labeled sample Immediately perform follow-up detection or store briefly at 4°C for each use.
[0066] 2. Before loading the sample, inject the hydrophobic agent for more than 15 minutes, and then rinse the pipeline with ultrapure water for 15 minutes. First introduce the negative control sample (1×PBS only contains the same concentration of fluorescent antibody as the cell sample), and adjust the flow rate to 0.2-0.8 μL / min, and observe the real-time flow state of the liquid flow through the CCD imaging of the detection device to ensure the stability of the liquid flow. In the real-time signal acquisition software, set the fluorescence signal acqu...
Embodiment 3
[0073] The method of "microfluidic chip-based single cell counting" of the present invention is used to measure the concentration of human lymphocyte standard substance as an example of counting. The specific steps are:
[0074] 1. Sample preparation:
[0075] Configure negative control tubes and sample tubes. Add 1×PBS buffer to the negative control tube, and add a final concentration of 1×CD4-PE antibody. In the sample tube, dissolve the human lymphocytes with 1×PBS buffer, add the final concentration of 1×CD4-PE antibody, and react for 30 minutes at room temperature in the dark.
[0076] 2. Adjustment of liquid flow system
[0077] A PBS solution containing 2% BSA (passing time is about 15 min) and double distilled water (passing time is 15 min) are successively passed through the microfluidic chip through a pressure pump, and then a negative control solution is introduced. According to the CCD of the imaging device to observe the movement state of the liquid circuit, t...
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