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A separation method, granulocyte technology, applied in the field of granulocyte separation, can solve problems such as low purity, red blood cell adhesion, and short shelf life of reagents, and achieve the effect of improving purity and reducing costs
Pending Publication Date: 2021-08-13
上海赛笠生物科技有限公司
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Problems solved by technology
Direct separation of percoll usually results in red blood cell adhesion, usually the purity is not high, and manual calculation of the proportion of percoll is relatively cumbersome
However, the magnetic bead sorting method has the problems of high cost and short shelf life of reagents.
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Embodiment 1
[0022] A new method for separating granulocytes, specifically comprising the following steps:
[0023] Step 1, configuring percoll with a density of 1.090g / mL;
[0024] Step 2, using commercialized ficoll with a density of 1.077g / mL to perform gradient density centrifugation on peripheral blood;
[0025] Step 3, removing the centrifuged supernatant, middle buffy coat and ficoll layer, leaving the bottom red blood cell mixture;
[0026] Step 4, wash the mixture once with PBS solution to remove the ficoll;
[0027] Step 5. Slowly add the washed cell mixture into the prepared 1.090g / mL percoll, and centrifuge at 400g for 20min;
[0028] Step 6, take out the middle layer after centrifugation;
[0029] Step 7, using the erythrocyte lysate to lyse the erythrocytes mixed in the centrifuged middle layer;
[0030] Step 8, adding PBS solution, washing away the red blood cell lysate, and the harvested cells are granulocytes.
Embodiment 2
[0032] A new granulocyte separation method specifically includes the following steps, and the method is verified by a verification test, and the verification test results are as follows: Figure 1-Figure 3 Shown:
[0033] Step 1, configuring percoll with a density of 1.090g / mL;
[0034] Step 2, using commercialized ficoll with a density of 1.077g / mL to perform gradient density centrifugation on peripheral blood;
[0035] Step 3, removing the centrifuged supernatant, middle buffy coat and ficoll layer, leaving the bottom red blood cell mixture;
[0036] Step 4, wash the mixture once with PBS solution to remove the ficoll;
[0037] Step 5. Slowly add the washed cell mixture into the prepared 1.090g / mL percoll, and centrifuge at 400g for 20min;
[0038] Step 6, take out the middle layer after centrifugation;
[0039] Step 7, using the erythrocyte lysate to lyse the erythrocytes mixed in the centrifuged middle layer;
[0040] Step 8, adding PBS solution, washing away the red ...
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Abstract
The invention provides a novel granulocyteseparation method. The novel granulocyteseparation method specifically comprises the following steps of: preparing percoll with the density of 1.090 g / mL; performing gradient density centrifugation on peripheral blood by using ficoll with the commercialized density of 1.077 g / mL; removing a supernatant, a middle albuginea layer and a ficoll layer, and leaving a lowermost erythrocyte mixture; cleaning the mixture once by using a PBS solution, and removing ficoll in the mixture; slowly adding the washed cell mixture into prepared percoll with the density of 1.090 g / mL, and carrying out centrifugation on 400g of the mixture for 20 minutes; taking out the middle layer after centrifugation is finished; carrying out splitting decomposition treatment on erythrocytes mixed in the middle layer after centrifugation by using an erythrocyte splitting decomposition solution; and adding a PBS, and washing off erythrocyte lysate to obtain the granulocyte. According to the invention, the purity of granulocytes can be greatly improved; and compared with a magnetic bead sorting method, the cost is reduced.
Description
technical field [0001] The invention belongs to the technical field of granulocyte separation, and in particular relates to a new method for granulocyte separation. Background technique [0002] According to the different morphology, leukocytes can be divided into granular type and non-granular type. Granulocytes are divided into neutrophils, eosinophils and basophils. The vast majority of granulocytes are neutrophils, which play chemotaxis, phagocytosis and bactericidal functions in the non-specific cellular immune system of the blood. Eosinophils play an important role in killing parasites such as parasitic nematodes. The cytoplasm of basophils contains a variable number of granules. Granules are rich in histamine, heparin, chondroitinsulfate, peroxidase, platelet activator and other substances. [0003] Existing methods for isolating granulocytes usually use peripheral blood percoll gradient density separation or magnetic bead sorting. Direct separation of percoll u...
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