Liver cancer detection kit based on liquid biopsy
A technology of liquid biopsy and reagent kit, which is applied in measuring devices, instruments, disease diagnosis, etc. It can solve the problems of weak fluorescence intensity, difficulty in distinguishing, and inconsistency, and achieve the effect of strong fluorescence, good staining effect, and enhanced staining effect
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Embodiment 1
[0055] Example 1 enrichment of target cells in peripheral blood
[0056] (1) Centrifuge peripheral blood to remove plasma protein: 8.5mL peripheral blood was centrifuged at 800g in a horizontal centrifuge for 7min at room temperature, and the supernatant was discarded.
[0057] (2) Add 5-6 mL of PBS buffer solution and 3 mL of lymphocyte separation solution to the plasma in step (1), centrifuge at 450 g in a centrifuge for 7 minutes at room temperature. After centrifugation, it is divided into three layers. The red bottom layer is the erythrocyte layer, the slightly white middle layer is mainly white blood cells and CTC, etc., and the yellow upper layer is plasma. Absorb all the liquid above the erythrocyte layer and remove the bottom erythrocyte layer.
[0058] (3) Add 200 mL of immunomagnetic beads with CD45 antibody coupled to the surface dropwise in step (2), and incubate on a horizontal shaker to obtain a suspension. At room temperature, shake horizontally for 20 minutes...
Embodiment 2
[0060] Example 2 Fluorescent staining of enriched target cells
[0061] (1) Enhanced staining pretreatment: Add 2 μL of staining enhancement solution to about 50 μL of enriched target cells, and let stand at room temperature for 10 min. The staining enhancing solution is a PBS buffer solution of SDS or Triton X-100, and the SDS concentration is 0.1 mg / mL.
[0062] (2) Cell surface staining: Dilute 1 μL each of CD45-Alexa 594 and ASGPR1-Alexa 488 with 198 μL of PBS buffer, add to the cell suspension after pretreatment in step (1), and incubate in the dark for 20 minutes. After incubation, add PBS buffer to wash the cell liquid, centrifuge at 950g for 4min, and remove the supernatant to 100μL.
[0063] (3) Cell fixation: transfer and smear the cells in step (2) onto a glass slide, then add the fixative paraformaldehyde, fix for 10 min, and wash the slide twice with PBS, 5 min each time.
[0064] (4) Cell membrane rupture: After the cells were fixed, 200 μL of cell membrane rup...
Embodiment 3
[0068] Example 3 Fluorescence staining detection of Hep3B human liver cancer cell line
[0069] Hep3B human liver cancer cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were enzymatically digested and 105 Cells, approximately 50 μL, were subjected to cell fluorescence staining and fluorescence microscope examination according to the steps in Example 2. Microscopic examination conditions are as follows: when the excitation light wavelength is 591nm, Alexa594 emits 618nm red light, and the exposure time is 100ms; when the excitation light wavelength is 650nm, CY5 emits 670nm far-red light (invisible to the naked eye, and the microscope scanning is assigned purple). The time is 100ms; when the excitation light wavelength is 499nm, Alexa488 emits 519nm green light, and the exposure time is 100ms; when the excitation light wavelength is 345nm, DAPI emits 455nm blue light, and the exposure time is 10-20ms, the results are as follows figure 1 shown.
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