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CN113151169A - Method for separating natural killer cells based on magnetic bead positive selection strategy - Google Patents

Method for separating natural killer cells based on magnetic bead positive selection strategy Download PDF

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CN113151169A
CN113151169A CN202110527157.XA CN202110527157A CN113151169A CN 113151169 A CN113151169 A CN 113151169A CN 202110527157 A CN202110527157 A CN 202110527157A CN 113151169 A CN113151169 A CN 113151169A
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向洋
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Shanghai Saili Biotechnology Co ltd
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Abstract

本发明提供了一种基于磁珠阳选策略分离自然杀伤细胞的方法，具体包括以下内容；使用磁珠分选缓冲液重悬外周血单个核细胞；加入CD14antibodycocktail并混匀孵育；涡旋后加入Rapidspheres并混匀孵育；插入磁铁内孵育；得到上清细胞；重悬上清细胞；加入CD3antibodycocktail混匀孵育；涡旋后加入Rapidspheres并混匀孵育；插入磁铁内孵育，得到上清细胞；加入CD56antibodycocktail并混匀孵育；涡旋后加入Rapidspheres并混匀孵育；插入磁铁内孵育；倒出上清，获得自然杀伤细胞。

Figure 202110527157

The invention provides a method for separating natural killer cells based on a magnetic bead positive selection strategy, which specifically includes the following contents: resuspending peripheral blood mononuclear cells with a magnetic bead sorting buffer; adding CD14antibodycocktail and mixing and incubating; adding after vortexing Rapidspheres and mix and incubate; insert into magnet and incubate; get supernatant cells; resuspend supernatant cells; add CD3antibodycocktail and mix and incubate; vortex, add Rapidspheres and mix and incubate; insert into magnet and incubate to obtain supernatant cells; add CD56antibodycocktail Mix and incubate; vortex, add Rapidspheres and mix and incubate; insert into a magnet and incubate; pour off the supernatant to obtain natural killer cells.

Figure 202110527157

Description

Method for separating natural killer cells based on magnetic bead positive selection strategy
Technical Field
The invention belongs to the technical field of NK cell magnetic bead sorting, and particularly relates to a method for separating natural killer cells based on a magnetic bead positive sorting strategy.
Background
Immunomagnetic bead sorting is a very effective means of cell separation. Generally, a positive sorting strategy for labeling cells of interest, and a negative sorting strategy for labeling non-cells of interest are classified. Negative cell sorting, although specific, is not high in yield and has a low sample utilization rate. Positive cell sorting strategy is not good for cell specificity due to the use of single marker sorting, and if CD56 is used as the marker for positive sorting of cells, it will contain a large proportion of monocytes and NKT cells, and will not be able to obtain ideal natural killer cells.
Disclosure of Invention
The invention aims to provide a method for separating natural killer cells based on a magnetic bead positive selection strategy, so as to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme: a method for separating natural killer cells based on a magnetic bead positive selection strategy specifically comprises the following steps:
step 1, resuspending peripheral blood mononuclear cells into 14ml round bottom tubes using magnetic bead sorting buffer to a final concentration of 1 x 108cells/mL；
Step 2, adding 100ul of CD14antibody cocktail into each ml of cells, uniformly mixing and incubating for 10 min;
step 3, vortexing the magnetic beads for 30s, adding 100ul of Rapidpheres into each ml of cells, uniformly mixing and incubating for 3 min;
step 4, supplementing the volume to 10ml and inserting the sample into a magnet for incubation for 3 min;
step 5, slowly taking up the magnet to pour out the supernatant, thereby realizing the separation of the supernatant cells and the magnetic beads and obtaining the supernatant cells;
step 6, resuspending the supernatant cells from step 5 in a 14ml round bottom tube using magnetic bead sorting buffer to a final concentration of 1 x 108cells/mL；
Step 7, adding 100ul of CD3antibody cocktail to each ml of cells, mixing uniformly and incubating for 3 min;
step 8, vortexing the magnetic beads for 30s, adding 60ul of Rapidpheres into each ml of cells, uniformly mixing and incubating for 3 min;
step 9, supplementing the volume to 10ml and inserting the sample into a magnet for incubation for 3 min;
step 10, slowly taking up the magnet to pour out the supernatant, thereby obtaining supernatant cells;
step 11, resuspending the supernatant cells from step 10 in magnetic bead sorting buffer into 14ml round-bottomed tubes to a final concentration of 1 x 108cells/mL；
Step 12, adding 100ul of CD56antibody cocktail to each ml of cells, mixing uniformly and incubating for 3 min;
step 13, vortexing the magnetic beads for 30s, adding 100ul of Rapidpheres into each ml of cells, uniformly mixing and incubating for 3 min;
step 14, supplementing the volume to 10ml and inserting the sample into a magnet for incubation for 3 min;
step 15, slowly taking up the magnet, pouring out supernatant, and resuspending the magnetic beads by using 10ml of buffer solution;
and step 16, repeating the step 14 and the step 15 twice to obtain the natural killer cells.
Preferably, the vortex speed in step 3, step 8 and step 13 is 3000 rpm.
Preferably, after the supernatant is poured out in the step 5, the magnetic beads are resuspended in 10ml of buffer solution, and then the two times of incubation in the magnet are combined for 3min, and the supernatant and the magnetic beads are separated, so that the mononuclear cells can be obtained.
Preferably, after the supernatant is poured out in the step 10, the magnetic beads are resuspended in 10ml of buffer solution, and then the two times of incubation in the magnet are combined for 3min, and the supernatant and the magnetic beads are separated, so that the T lymphocytes can be obtained.
Preferably, steps 1-16 are performed in a class II biosafety cabinet, and strict adherence to sterile procedures is maintained.
The invention has the technical effects and advantages that: the invention can obtain high-specificity natural killer cells with higher yield through reasonable separation steps, and can also obtain certain T lymphocytes and mononuclear cells as byproducts, thereby improving the utilization rate of peripheral blood mononuclear cells.
Drawings
FIG. 1 is a forward side scatter plot of a test sample;
FIG. 2 is a double fluorescent dot plot of CD3/CD56 in a cell sample (NK cells in the second quadrant proportion).
Detailed Description
Example 1
A method for separating natural killer cells based on a magnetic bead positive selection strategy specifically comprises the following steps:
step 1, resuspending peripheral blood mononuclear cells into 14ml round bottom tubes using magnetic bead sorting buffer to a final concentration of 1 x 108cells/mL；
Step 2, adding 100ul of CD14antibody cocktail into each ml of cells, uniformly mixing and incubating for 10 min;
step 3, vortexing the magnetic beads for 30s, adding 100ul of Rapidpheres into each ml of cells, uniformly mixing and incubating for 3 min;
step 4, supplementing the volume to 10ml and inserting the sample into a magnet for incubation for 3 min;
step 5, slowly taking up the magnet to pour out the supernatant, thereby realizing the separation of the supernatant cells and the magnetic beads and obtaining the supernatant cells;
step 6, resuspending the supernatant cells from step 5 in a 14ml round bottom tube using magnetic bead sorting buffer to a final concentration of 1 x 108cells/mL；
Step 7, adding 100ul of CD3antibody cocktail to each ml of cells, mixing uniformly and incubating for 3 min;
step 8, vortexing the magnetic beads for 30s, adding 60ul of Rapidpheres into each ml of cells, uniformly mixing and incubating for 3 min;
step 9, supplementing the volume to 10ml and inserting the sample into a magnet for incubation for 3 min;
step 10, slowly taking up the magnet to pour out the supernatant, thereby obtaining supernatant cells;
step 11, resuspending the supernatant cells from step 10 in magnetic bead sorting buffer into 14ml round-bottomed tubes to a final concentration of 1 x 108cells/mL；
Step 12, adding 100ul of CD56antibody cocktail to each ml of cells, mixing uniformly and incubating for 3 min;
step 13, vortexing the magnetic beads for 30s, adding 100ul of Rapidpheres into each ml of cells, uniformly mixing and incubating for 3 min;
step 14, supplementing the volume to 10ml and inserting the sample into a magnet for incubation for 3 min;
step 15, slowly taking up the magnet, pouring out supernatant, and resuspending the magnetic beads by using 10ml of buffer solution;
and step 16, repeating the step 14 and the step 15 twice to obtain the natural killer cells.
Example 2
A method for separating natural killer cells based on a magnetic bead positive selection strategy specifically comprises the following steps, and detection is carried out on a separation result, wherein the detection result is shown in figures 1 and 2:
step 1, resuspending peripheral blood mononuclear cells into 14ml round bottom tubes using magnetic bead sorting buffer to a final concentration of 1 x 108cells/mL；
Step 2, adding 100ul of CD14antibody cocktail into each ml of cells, uniformly mixing and incubating for 10 min;
step 3, vortexing the magnetic beads for 30s, adding 100ul of Rapidpheres into each ml of cells, uniformly mixing and incubating for 3 min;
step 4, supplementing the volume to 10ml and inserting the sample into a magnet for incubation for 3 min;
step 5, slowly taking up the magnet to pour out the supernatant, thereby realizing the separation of the supernatant cells and the magnetic beads and obtaining the supernatant cells;
step 6, resuspending the supernatant cells from step 5 in a 14ml round bottom tube using magnetic bead sorting buffer to a final concentration of 1 x 108cells/mL；
Step 7, adding 100ul of CD3antibody cocktail to each ml of cells, mixing uniformly and incubating for 3 min;
step 8, vortexing the magnetic beads for 30s, adding 60ul of Rapidpheres into each ml of cells, uniformly mixing and incubating for 3 min;
step 9, supplementing the volume to 10ml and inserting the sample into a magnet for incubation for 3 min;
step 10, slowly taking up the magnet to pour out the supernatant, thereby obtaining supernatant cells;
step 11, resuspending the supernatant cells from step 10 in magnetic bead sorting buffer into 14ml round-bottomed tubes to a final concentration of 1 x 108cells/mL；
Step 12, adding 100ul of CD56antibody cocktail to each ml of cells, mixing uniformly and incubating for 3 min;
step 13, vortexing the magnetic beads for 30s, adding 100ul of Rapidpheres into each ml of cells, uniformly mixing and incubating for 3 min;
step 14, supplementing the volume to 10ml and inserting the sample into a magnet for incubation for 3 min;
step 15, slowly taking up the magnet, pouring out supernatant, and resuspending the magnetic beads by using 10ml of buffer solution;
and step 16, repeating the step 14 and the step 15 twice to obtain the natural killer cells.
Preferably, the vortex speed in step 3, step 8 and step 13 is 3000 rpm.
Preferably, after the supernatant is poured out in the step 5, the magnetic beads are resuspended in 10ml of buffer solution, and then the two times of incubation in the magnet are combined for 3min, and the supernatant and the magnetic beads are separated, so that the mononuclear cells can be obtained.
Preferably, after the supernatant is poured out in the step 10, the magnetic beads are resuspended in 10ml of buffer solution, and then the two times of incubation in the magnet are combined for 3min, and the supernatant and the magnetic beads are separated, so that the T lymphocytes can be obtained.
Preferably, steps 1-16 are performed in a class II biosafety cabinet, and strict adherence to sterile procedures is maintained.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments or portions thereof without departing from the spirit and scope of the invention.

Claims (5)

1.一种基于磁珠阳选策略分离自然杀伤细胞的方法，其特征在于：具体包括以下步骤，1. a method for separating natural killer cells based on magnetic bead positive selection strategy, is characterized in that: specifically comprise the following steps, 步骤1、使用磁珠分选缓冲液重悬外周血单个核细胞至14ml圆底试管中使其终浓度为1*108cells/mL；Step 1. Resuspend peripheral blood mononuclear cells in a 14ml round-bottom test tube with magnetic bead sorting buffer to make the final concentration 1*10 8 cells/mL; 步骤2、每毫升细胞加入100ul CD 14antibody cocktail并混匀孵育10min；Step 2. Add 100ul CD 14antibody cocktail per ml of cells and mix and incubate for 10min; 步骤3、涡旋磁珠30s，每毫升细胞加入100ul Rapidspheres并混匀孵育3min；Step 3. Vortex the magnetic beads for 30s, add 100ul Rapidspheres per ml of cells, mix and incubate for 3min; 步骤4、将体积补到10ml并将样本插入磁铁内孵育3min；Step 4. Make up the volume to 10ml and insert the sample into the magnet and incubate for 3min; 步骤5、缓缓拿起磁铁倒出上清，从而实现上清细胞和磁珠的分离，得到上清细胞；Step 5. Slowly pick up the magnet and pour out the supernatant, so as to realize the separation of the supernatant cells and the magnetic beads, and obtain the supernatant cells; 步骤6、使用磁珠分选缓冲液重悬步骤5所得上清细胞至14ml圆底试管中使其终浓度为1*108cells/mL；Step 6. Use magnetic bead sorting buffer to resuspend the supernatant cells obtained in Step 5 into a 14ml round-bottom test tube to make the final concentration 1*10 8 cells/mL; 步骤7、每毫升细胞加入100ul CD3 antibody cocktail并混匀孵育3min；Step 7. Add 100ul CD3 antibody cocktail per milliliter of cells and mix and incubate for 3 minutes; 步骤8、涡旋磁珠30s，每毫升细胞加入60ul Rapidspheres并混匀孵育3min；Step 8. Vortex the magnetic beads for 30s, add 60ul Rapidspheres per ml of cells, mix and incubate for 3min; 步骤9、.将体积补到10ml并将样本插入磁铁内孵育3min；Step 9. Make up the volume to 10ml and insert the sample into the magnet and incubate for 3min; 步骤10、缓缓拿起磁铁倒出上清，从而得到上清细胞；Step 10. Slowly pick up the magnet and pour out the supernatant to obtain supernatant cells; 步骤11、使用磁珠分选缓冲液重悬步骤10所得上清细胞至14ml圆底试管中使其终浓度为1*108cells/mL；Step 11. Use magnetic bead sorting buffer to resuspend the supernatant cells obtained in Step 10 into a 14ml round-bottom test tube to make the final concentration 1*10 8 cells/mL; 步骤12、每毫升细胞加入100ul CD56 antibody cocktail并混匀孵育3min；Step 12. Add 100ul CD56 antibody cocktail per milliliter of cells, mix and incubate for 3 minutes; 步骤13、涡旋磁珠30s，每毫升细胞加入100ul Rapidspheres并混匀孵育3min；Step 13. Vortex the magnetic beads for 30s, add 100ul Rapidspheres per ml of cells, mix and incubate for 3min; 步骤14、将体积补到10ml并将样本插入磁铁内孵育3min；Step 14. Make up the volume to 10ml and insert the sample into the magnet and incubate for 3min; 步骤15、缓缓拿起磁铁倒出上清，并用10ml缓冲液重悬磁珠；Step 15. Slowly pick up the magnet and pour out the supernatant, and resuspend the magnetic beads with 10ml buffer; 步骤16、将步骤14和步骤15重复两次即可获得自然杀伤细胞。Step 16: Repeat steps 14 and 15 twice to obtain natural killer cells. 2.根据权利要求1所述的一种基于磁珠阳选策略分离自然杀伤细胞的方法，其特征在于：所述步骤3、步骤8和步骤13中的涡旋速度为3000rpm。2 . The method for separating natural killer cells based on a magnetic bead positive selection strategy according to claim 1 , wherein the vortex speed in the steps 3, 8 and 13 is 3000 rpm. 3 . 3.根据权利要求1所述的一种基于磁珠阳选策略分离自然杀伤细胞的方法，其特征在于：所述步骤5中倒出上清后，并用10ml缓冲液重悬磁珠，再结合两次磁铁内孵育3min、分离上清和磁珠，即可获得单核细胞。3. a kind of method for separating natural killer cells based on magnetic bead positive selection strategy according to claim 1, is characterized in that: after pouring out supernatant in described step 5, and resuspend magnetic beads with 10ml buffer solution, then combine Mononuclear cells can be obtained by incubating twice in a magnet for 3 min, separating the supernatant and magnetic beads. 4.根据权利要求1所述的一种基于磁珠阳选策略分离自然杀伤细胞的方法，其特征在于：所述步骤10中倒出上清后，并用10ml缓冲液重悬磁珠，再结合两次磁铁内孵育3min、分离上清和磁珠，即可获得T淋巴细胞。4. a kind of method for separating natural killer cells based on magnetic bead positive selection strategy according to claim 1, is characterized in that: after pouring out supernatant in described step 10, and resuspend magnetic beads with 10ml buffer solution, then combine T lymphocytes were obtained by incubating twice in a magnet for 3 min, separating the supernatant and magnetic beads. 5.根据权利要求1所述的一种基于磁珠阳选策略分离自然杀伤细胞的方法，其特征在于：所述步骤1-步骤16须在II级生物安全柜内操作，并严格遵守无菌操作。5. a kind of method for separating natural killer cells based on magnetic bead positive selection strategy according to claim 1, is characterized in that: described step 1-step 16 must be operated in II-level biological safety cabinet, and strictly abide by aseptic operate.
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