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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cells
supernatant
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magnetic bead
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向洋
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Shanghai Saili Biotechnology Co ltd
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells
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Abstract

The invention provides a method for separating natural killer cells based on a magnetic bead positive selection strategy, which specifically comprises the following steps; resuspending peripheral blood mononuclear cells using a magnetic bead sorting buffer; adding CD14 antibodycoccus trail, mixing uniformly and incubating; adding Rapidstepheres after vortexing, and uniformly mixing and incubating; inserting into a magnet for incubation; obtaining supernatant cells; resuspending the supernatant cells; adding CD3 antibodyycocktail, mixing uniformly and incubating; adding Rapidstepheres after vortexing, and uniformly mixing and incubating; inserting the cells into a magnet for incubation to obtain supernatant cells; adding CD56 antibiomycocktail, mixing uniformly and incubating; adding Rapidstepheres after vortexing, and uniformly mixing and incubating; inserting into a magnet for incubation; the supernatant was decanted to obtain natural killer cells.

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. A method for separating natural killer cells based on a magnetic bead positive selection strategy is characterized by comprising the following steps: the method specifically comprises the following steps of,
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.
2. The method for separating natural killer cells based on the magnetic bead positive selection strategy as claimed in claim 1, wherein: the vortex speed in step 3, step 8 and step 13 was 3000 rpm.
3. The method for separating natural killer cells based on the magnetic bead positive selection strategy as claimed in claim 1, wherein: and (5) pouring out the supernatant in the step (5), re-suspending the magnetic beads by using 10ml of buffer solution, then combining the two times of magnet incubation for 3min, and separating the supernatant and the magnetic beads to obtain the mononuclear cells.
4. The method for separating natural killer cells based on the magnetic bead positive selection strategy as claimed in claim 1, wherein: and (3) after the supernatant is poured out in the step 10, resuspending the magnetic beads by using 10ml of buffer solution, then combining the two times of magnet incubation for 3min, and separating the supernatant and the magnetic beads to obtain the T lymphocytes.
5. The method for separating natural killer cells based on the magnetic bead positive selection strategy as claimed in claim 1, wherein: the steps 1-16 must be performed in a class II biosafety cabinet, and strict adherence to aseptic procedures.
CN202110527157.XA 2021-05-14 2021-05-14 Method for separating natural killer cells based on magnetic bead positive selection strategy Pending CN113151169A (en)

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CA2785627A1 (en) * 2009-12-29 2011-07-07 Gamida-Cell Ltd. Methods for enhancing natural killer cell proliferation and activity
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