Preparation and identification method of human leukemia cell cytoplast

A leukemia cell and cytoplasmic technology, applied in the field of preparation and identification of human leukemia cell cytoplasm, can solve the problem that the cytoplasm cannot be directly stained, the yield of cytoplasm is not high, and the cytoplasm cannot be used for follow-up research and other problems, to achieve the effect of inhibiting mitosis and increasing the rate of denucleation

Inactive Publication Date: 2012-06-13
AFFILIATED HOSPITAL OF ZUNYI MEDICAL COLLEGE
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Problems solved by technology

[0007] The technical problem that the present invention solves is Summary of the invention: the preparation and identification of existing human leukemia cell cytoplasts have a low yield of cytoplasts...

Method used

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Embodiment 1

[0017] The preparation method of human leukemia cell cytoplasm of the present invention specifically comprises the following steps:

[0018] First, take human leukemia HL-60 cells in the logarithmic growth phase, centrifuge at 1000 rpm for 5 minutes, remove the supernatant, suspend the precipitate with 1ml of medium, take 3ml of 42% Percoll solution in a 15ml centrifuge tube, and suspend the above cells Carefully spread the solution on the surface of Percoll solution, centrifuge at 1500 rpm for 30 minutes at 20°C, remove the buffy coat layer, and suspend the precipitate to 1ml in serum-free DMEM, which is purified HL-60 cells;

[0019] Second, the above-mentioned purified cell suspension (5×10 7 cells / ml) added to CB (final concentration 10 μg / ml)

[0020] and colchicine (final concentration: 5 μg / ml), and set only CB (final concentration: 10 μg / ml) group as a control, and incubate at 37°C for 30 minutes. Centrifuge at 1000 rpm for 5 minutes, remove the supernatant and leave...

Embodiment 2

[0024] The identification method of human leukemia cell cytoplasm of the present invention specifically comprises the following steps:

[0025] Fluorescent double staining to identify cytoplasts: adjust the volume of the above-mentioned purified cytoplast suspension to 1ml, add DNA fluorescent dye 4,6-diamidino-2-phenylindole dihydrochloride (4′,6 -diamidino-2-phenylindole, dihydrochloride, DAPI) stock solution (1mg / ml) 50μl (final concentration 50μg / ml), then add cytoplasmic protein fluorescent dye carboxyfluorescein diacetate succinimidyl ester (5, 6-carboxyflu-orescein diacetate succinimidyl ester, CFSE) stock solution (5 μmol / ml) 1 μl (final concentration 5nmol / ml), mix well, incubate at 37°C in the dark for 20 minutes, centrifuge at 10°C, 2 350 rpm for 12 Minutes, remove the supernatant, add 5ml PBS to suspend, centrifuge at 10°C, 2350 rpm for 12 minutes, remove the supernatant, repeat the same method twice, suspend the sediment with an appropriate amount of PBS, transfer...

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Abstract

The invention discloses a preparation and identification method of a human leukemia cell cytoplast. The method comprises the following steps of: treating a purified human leukemia HL-60 cell with a cytochalasin and colchicine, centrifugally denucleating at the temperature of 34 DEG C, collecting a cytoplast component, and purifying with a Percoll density gradient centrifugation method to obtain apurified cytoplast; and identifying the cytoplast with DAPI (4',6-diamidino-2-phenylindole) and CFSE (5,6-carboxyflu-orescein diacetate succinimidyl ester) fluorescent double staining. Due to the adoption of the method, the denucleating rate of a non-adherent human leukemia HL-60 cell can be over 90 percent, the purity of purified cytoplasts is over 95 percent, the quantity of cytoplasts which are more than or equal to 5 mum in diameter is up to 81 percent, and a cytoplast which is chromophilic with a CFSE fluorescent probe can be directly applied to subsequent researches; an HL-60 cell cytoplast obtained with the method can be widely applied to researches of leukemia cell cytoplast components as well as metabolism, functions, cell reconstruction, cell differentiation, apoptosis and the like thereof; and the method is suitable for preparing and identifying all in-vitro non-adherent tumor cell cytoplasts.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for preparing and identifying human leukemia cell cytoplasm. Background technique [0002] Cytoplasm plays a decisive role in the process of cell differentiation. Cytoplast is an enucleated cell, which is a good biological model for studying mitochondrial DNA, plasma membrane components and functions, cell apoptosis, cell differentiation, etc. Cytoplasmic replacement is essential for cellular remodeling. The preparation method of cytoplasm is divided into two kinds: invasive denucleation and non-invasive denucleation. The former adopts micromanipulation mechanical enucleation, which is suitable for larger cells; the latter is in cytochalasin (Cytochalasin B, CB) mediated denucleation by centrifugation, suitable for smaller cells. The CB-mediated denucleation technique has been commonly used for the preparation of large quantities of cytoplasm. However, for the...

Claims

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Application Information

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IPC IPC(8): C12N5/09C12Q1/02G01N21/64
Inventor 陈代雄
Owner AFFILIATED HOSPITAL OF ZUNYI MEDICAL COLLEGE
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