CN113755434B - Leukocyte separation liquid and leukocyte separation method - Google Patents
Leukocyte separation liquid and leukocyte separation method Download PDFInfo
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- CN113755434B CN113755434B CN202111082421.XA CN202111082421A CN113755434B CN 113755434 B CN113755434 B CN 113755434B CN 202111082421 A CN202111082421 A CN 202111082421A CN 113755434 B CN113755434 B CN 113755434B
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Abstract
The invention discloses a leukocyte separation liquid and a leukocyte separation method, which relate to the field of cell engineering, wherein the leukocyte separation liquid comprises the following components in parts by mass: 20-24 parts of aspirin, 8-12 parts of sodium citrate, 16-20 parts of polydiallyl dimethyl ammonium chloride, 46-50 parts of ammonium chloride, 6-10 parts of potassium bicarbonate and 0.6-1.2 parts of EDTA; the formula of the separating liquid can not only enable anticoagulation and erythrocyte division to be carried out synchronously, but also improve the separating efficiency; and the separation rate is high, and the separation effect is good.
Description
Technical Field
The invention relates to the field of cell engineering, in particular to a leukocyte separation liquid and a leukocyte separation method.
Background
The ratio of red blood cells to white blood cells in blood is about 600-1000: 1, the specific gravity of the two materials is different, the sedimentation speed is different, and the two materials are separated by two methods; the white blood cell separation in the market usually comprises timely anticoagulation after blood collection, and heparin anticoagulation is usually selected, wherein heparin can prevent prothrombin from being converted into thrombin, so that fibrinogen is inhibited from forming fibrin, and blood coagulation is prevented. Standing the anticoagulated test tube upright for 30-60 min at room temperature, separating the blood into three obvious layers, wherein the upper layer is light yellow blood plasma, the bottom layer is red blood cells, the gray layer closely attached to the upper surface of the red blood cell layer is white blood cells, slightly sucking to obtain a cell group rich in white blood cells, adding a small amount of distilled water or Gey solution containing ammonium chloride after centrifugal washing, performing short-time hypotonic treatment to crack the red blood cells, and repeatedly washing to obtain a white blood cell suspension with higher purity; the method in the prior art has the defects of complicated operation, two steps of operations for coagulation and separation, poor separation effect of white blood cells and low separation rate.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide a white blood cell separating liquid and a white blood cell separating method, wherein the formula of the separating liquid can synchronously perform anticoagulation and erythrocyte division, and the obtained white blood cell separating rate is high and the separating effect is good.
In order to achieve the above object, the present invention adopts the following technical scheme:
a leukocyte separation liquid comprises the following components in parts by mass: 20-24 parts of aspirin, 8-12 parts of sodium citrate, 16-20 parts of polydiallyl dimethyl ammonium chloride, 46-50 parts of ammonium chloride, 6-10 parts of potassium bicarbonate and 0.6-1.2 parts of EDTA.
The formula of the leukocyte separation liquid comprises the following components in parts by mass: 22.3 parts of aspirin, 9.4 parts of sodium citrate, 18.6 parts of polydiallyl dimethyl ammonium chloride, 48.4 parts of ammonium chloride, 8.5 parts of potassium bicarbonate and 0.8 part of EDTA.
The formula of the white blood cell separating liquid is as follows: 22.3g of aspirin, 9.4g of sodium citrate, 18.6g of polydiallyl dimethyl ammonium chloride, 48.4g of ammonium chloride, 8.5g of potassium bicarbonate and 0.8g of EDTA0.8g, and adding water to 1L.
A method of leukocyte separation comprising the steps of:
step one, preparing a leukocyte separation liquid, and pouring the separation liquid into a centrifuge tube;
the formula comprises the following components in parts by mass: 20-24 parts of aspirin, 8-12 parts of sodium citrate, 16-20 parts of polydiallyl dimethyl ammonium chloride, 46-50 parts of ammonium chloride, 6-10 parts of potassium bicarbonate and 0.6-1.2 parts of EDTA;
step two, after blood is collected, fresh blood is put into a centrifuge tube with a white blood cell separating liquid poured in, and PBS with the same volume as the white blood cell separating liquid is added for dilution;
step three, centrifuging at room temperature;
taking out the centrifuge tube, sucking out a white layer, re-suspending in PBS with the volume of 4-6 times, and uniformly mixing;
step five, repeating the centrifugation at room temperature, absorbing and discarding the supernatant, adding the incomplete culture solution, and uniformly mixing;
step six, counting lymphocytes;
and step seven, diluting the cells and the plates according to the required concentration and experimental purposes.
In the above-described method for separating white blood cells, the incomplete culture solution is an incomplete DMEM culture solution.
In the above-mentioned method for separating white blood cells, the DMEM culture solution is composed of DMEM 12.67g, sodium bicarbonate 2.8g, double antibody solution 10ml, 100X L.G solution 10ml, hydrochloric acid is added until pH is 7.2-7.4, and ultrapure water is added to 1L.
In the fifth step, centrifugation at room temperature, supernatant suction, incomplete culture solution addition, and uniform mixing are repeated for two to three times.
The invention has the advantages that:
the formula of the separating liquid can synchronously perform anticoagulation and erythrocyte division, and the separating rate is high, so that the method has substantial progress compared with the existing white blood cell separating method;
the invention discovers that the separation rate can be improved by combining aspirin, sodium citrate and polydiallyl dimethyl ammonium chloride.
Detailed Description
The present invention will be specifically described with reference to the following specific examples.
A method of leukocyte separation comprising the steps of:
step one, preparing a leukocyte separation liquid, and pouring the separation liquid into a centrifuge tube;
the formula is as follows: 20-24 parts of aspirin, 8-12 parts of sodium citrate, 16-20 parts of polydiallyl dimethyl ammonium chloride, 46-50 parts of ammonium chloride, 6-10 parts of potassium bicarbonate and 0.6-1.2 parts of EDTA;
step two, after blood is collected, fresh blood is put into a centrifuge tube with a white blood cell separating liquid poured in, and PBS with the same volume as the white blood cell separating liquid is added for dilution;
step three, centrifuging at room temperature;
taking out the centrifuge tube, sucking out a white layer, re-suspending in PBS with the volume of 4-6 times, and uniformly mixing;
step five, repeating the centrifugation at room temperature, absorbing and discarding the supernatant, adding the incomplete culture solution, and uniformly mixing; as an example, the incomplete culture solution is an incomplete DMEM culture solution; the DMEM culture solution was DMEM (12.67 g), sodium bicarbonate (2.8 g), diabody solution (10 ml), and 100 x L.G solution (10 ml), hydrochloric acid was added until pH was 7.2-7.4, and ultrapure water was added to 1L. The incomplete culture solution is not limited, and any serum-free culture solution that can culture white blood cells can be used in the present invention. This set of actions is preferably repeated two to three times, but may also be repeated twice.
Step six, counting lymphocytes;
and step seven, diluting the cells and the plates according to the required concentration and experimental purposes.
The following experiments were performed to isolate leukocytes, and the beneficial effects of the invention were demonstrated by testing the leukocyte isolation rate:
example 1:
taking 20ml of blood in the same sample as a sample 1 and a sample 2 respectively;
the following experiment was performed with sample 1:
step one, preparing a leukocyte separation liquid, and pouring the separation liquid into a centrifuge tube;
the formula is as follows: 20g of aspirin, 8g of sodium citrate, 16g of polydiallyl dimethyl ammonium chloride, 46g of ammonium chloride, 6g of potassium bicarbonate, 0.6g of EDTA and adding water to 1L;
step two, after blood is collected, fresh blood is put into a centrifuge tube with a white blood cell separating liquid poured in, and PBS with the same volume as the white blood cell separating liquid is added for dilution;
step three, centrifuging at 2600rpm for 20 minutes at room temperature;
and step four, taking out the centrifuge tube, sucking out a white layer, re-suspending in PBS with the volume of 4-6 times, uniformly mixing, and counting by using a blood cell analyzer.
Immediately after anticoagulation with heparin with sample 2, counts were taken with a blood cell analyzer.
Example 2:
the formula is as follows: 22.3g of aspirin, 9.4g of sodium citrate, 18.6g of polydiallyl dimethyl ammonium chloride and 48.4g of ammonium chloride; 8.5g of potassium bicarbonate; EDTA0.8g, water to 1L;
the procedure is as in example 1.
Example 3:
the formula is as follows: 24g of aspirin, 12g of sodium citrate, 20g of polydiallyl dimethyl ammonium chloride and 50g of ammonium chloride; 10g of potassium bicarbonate; EDTA 1.2g, water to 1L;
the procedure is as in example 1.
Comparative example 1:
the formula is as follows: 9.4g of sodium citrate, 18.6g of polydiallyl dimethyl ammonium chloride and 48.4g of ammonium chloride; 8.5g of potassium bicarbonate; EDTA0.8g, water to 1L;
the procedure is as in example 1.
Comparative example 2:
the formula is as follows: 22.3g of aspirin, 18.6g of polydiallyl dimethyl ammonium chloride and 48.4g of ammonium chloride; 8.5g of potassium bicarbonate; EDTA0.8g, water to 1L;
the procedure is as in example 1.
Comparative example 3:
the formula is as follows: 22.3g of aspirin, 9.4g of sodium citrate and 48.4g of ammonium chloride; 8.5g of potassium bicarbonate; EDTA0.8g, water to 1L;
the procedure is as in example 1.
Separation rate = count value of sample 1/count value of sample 2.
The results are shown in Table 1:
TABLE 1
| Sample 1 count value 10 9 /L | Sample 2 count value 10 9 /L | Separation rate% | |
| Example 1 | 1.62 | 1.78 | 91 |
| Example 2 | 1.66 | 1.76 | 94 |
| Example 3 | 1.60 | 1.76 | 91 |
| Comparative example 1 | 1.43 | 1.80 | 79 |
| Comparative example 2 | 1.42 | 1.77 | 80 |
| Comparative example 3 | 1.34 | 1.78 | 75 |
From the results in table 1, it can be seen that: the formula of the invention has better separation rate, and aspirin, sodium citrate and ammonium chloride are lack of any one, so that the separation rate is low. The formula of the separating liquid can enable anticoagulation and erythrocyte division to be carried out synchronously, and the separating rate is high, so that the method has substantial progress compared with the existing white blood cell separating method.
The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be appreciated by persons skilled in the art that the above embodiments are not intended to limit the invention in any way, and that all technical solutions obtained by means of equivalent substitutions or equivalent transformations fall within the scope of the invention.
Claims (7)
1. The white blood cell separating liquid is characterized by comprising the following components in parts by weight: 20-24 parts of aspirin, 8-12 parts of sodium citrate, 16-20 parts of polydiallyl dimethyl ammonium chloride, 46-50 parts of ammonium chloride, 6-10 parts of potassium bicarbonate and 0.6-1.2 parts of EDTA.
2. The white blood cell separating liquid according to claim 1, wherein the formula comprises the following components in parts by mass: 22.3 parts of aspirin, 9.4 parts of sodium citrate, 18.6 parts of polydiallyl dimethyl ammonium chloride and 48.4 parts of ammonium chloride; 8.5 parts of potassium bicarbonate; EDTA0.8 parts.
3. The white blood cell separating fluid of claim 1, wherein the formulation is: 22.3g of aspirin, 9.4g of sodium citrate, 18.6g of polydiallyl dimethyl ammonium chloride and 48.4g of ammonium chloride; 8.5g of potassium bicarbonate; EDTA0.8g, water was added to 1L.
4. A method for separating white blood cells, comprising the steps of:
step one, preparing a leukocyte separation liquid, and pouring the separation liquid into a centrifuge tube;
the formula comprises the following components in parts by mass: 20-24 parts of aspirin, 8-12 parts of sodium citrate, 16-20 parts of polydiallyl dimethyl ammonium chloride, 46-50 parts of ammonium chloride, 6-10 parts of potassium bicarbonate and 0.6-1.2 parts of EDTA;
step two, after blood is collected, fresh blood is put into a centrifuge tube with a white blood cell separating liquid poured in, and PBS with the same volume as the white blood cell separating liquid is added for dilution;
step three, centrifuging at room temperature;
taking out the centrifuge tube, sucking out a white layer, re-suspending in PBS with the volume of 4-6 times, and uniformly mixing;
step five, repeating the centrifugation at room temperature, absorbing and discarding the supernatant, adding the incomplete culture solution, and uniformly mixing;
step six, counting lymphocytes;
and step seven, diluting the cells and the plates according to the required concentration and experimental purposes.
5. The method according to claim 4, wherein the incomplete culture solution is an incomplete DMEM culture solution.
6. A method for separating white blood cells according to claim 5, wherein the DMEM medium is DMEM 12.67g, sodium bicarbonate 2.8g, diabody solution 10ml,100 x L.G solution 10ml, hydrochloric acid is added until pH 7.2-7.4, and ultrapure water is added to 1L.
7. The method according to claim 4, wherein the centrifugation at room temperature, the suction of the supernatant, the addition of the incomplete culture solution, and the mixing are repeated two to three times.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| JPH05344883A (en) * | 1992-06-15 | 1993-12-27 | Suntory Ltd | Cloned human t-cell strain capable of mass production of il-5 and ifn-gamma |
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| US5702884A (en) * | 1996-03-12 | 1997-12-30 | Johnson & Johnson Clinical Diagnostics, Inc. | Whole blood sample preparation for polymerase chain reaction using ammonium chloride and a carboxylic acid or metal carboxylate for selective red blood cell lysis |
| WO2011014741A1 (en) * | 2009-07-31 | 2011-02-03 | Artemis Health, Inc. | Methods and compositions for cell stabilization |
| CN102703597B (en) * | 2012-06-20 | 2013-10-30 | 安徽信灵检验医学科技有限公司 | Blood leucocyte separating medium and preparation method and vacuum blood collection tube |
| CN105505868B (en) * | 2015-12-31 | 2019-02-15 | 北京弘润天源基因生物技术有限公司 | Separate the separation method of peripheral blood immunocyte |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH05344883A (en) * | 1992-06-15 | 1993-12-27 | Suntory Ltd | Cloned human t-cell strain capable of mass production of il-5 and ifn-gamma |
Non-Patent Citations (3)
| Title |
|---|
| Differential effects of aspirin and misoprostol on 15-hydroxyeicosatetraenoic acid generation by leukocytes from aspirinsensitive asthmatic patients;Kowalski等;《J ALLERGY CLIN IMMUNOL》;第505-512页 * |
| Maderna等.DIFFERENTIAL EFFECTS OF ASPIRIN AND INDOMETHACIN ON PLATELET AND LE'JKOCYTE THROMBOXANE A2 FORMATION .《ProstaglandinsL eukotrienesa nd Medicine》.1985,第18卷第379-391页. * |
| 阿司匹林对人外周血单个核细胞HLA-I类分子表达的影响;刘俊霞;李黎;章涛;;山东医药(第20期);第76-77页 * |
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