Macrophage culture method

Abstract

The invention discloses a macrophage culture method which comprises the following steps: step 1, suspending human monocyte leukemia THP-1 cells in a T25 culture bottle by using 6-8 mL of an RPMI-1640 complete medium, putting the T25 culture bottle in a 37 DEG C / 5% CO2 incubator for culture, adding 1-2 mL of a fresh medium into the bottle every 2-3 days, and performing passage when the cell density reaches 8 * 10 < 5 >-* 10 < 6 > cells / mL; step 2, adding phorbol ester PMA into the culture bottle when the cells grow to a proper concentration, enabling the final concentration to be 100ng / mL, lightly and uniformly mixing, and culturing in a CO2 incubator; step 3, stimulating phorbol ester PMA for 24 hours, and then observing the cell adherence condition; and step 4, detecting the expression condition of the macrophage marker CD11b by a real-time fluorescent quantitative nucleic acid amplification detection system qPCR, and if the expression of the CD11b is positive, identifying the macrophage as the macrophage. The macrophages cultured by the method are higher in purity and shorter in growth cycle, and experimental research is facilitated.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, and particularly to a method for culturing macrophages. Background Art

[0002] Macrophages are a type of white blood cell located in tissues, originating from monocytes, which in turn originate from precursor cells in the bone marrow. Both macrophages and monocytes are phagocytic cells and participate in non-specific and specific defenses in vertebrates. Their main function is to phagocytize cell debris and pathogens in the form of fixed cells or free cells, and activate lymphocytes or other immune cells to respond to pathogens. Macrophages belong to immune cells and have multiple functions. They are important objects for studying cell phagocytosis, cellular immunity, and molecular immunology. However, the macrophages cultured by existing macrophage culture methods have a low purity and a slow growth cycle, which is not conducive to experimental research. Therefore, it is necessary to improve the existing technology to solve the above problems. Summary of the Invention

[0003] The purpose of the present invention is to provide a method for culturing macrophages.

[0004] To solve the above technical problems, the present invention adopts the following technical solutions:

[0005] The present invention provides a method for culturing macrophages, which is characterized by including the following steps:

[0006] Step 1: Suspend human monocytic leukemia THP-1 cells in a T25 culture flask with 6 - 8 mL of RPMI-1640 complete medium, and place it in a 37°C / 5% CO2 incubator for culture. Add 1 - 2 mL of fresh medium to the flask every 2 - 3 days. Passage when the cell density reaches 8x105 - x106 cells / mL;

[0007] Step 2. When the cells grow to an appropriate concentration, add phorbol 12-myristate 13-acetate (PMA) to the culture flask to make its final concentration 100 ng / mL. Gently mix and then place it in a CO2 incubator for culture;

[0008] Step 3. Observe the cell adhesion situation 24 hours after stimulation with PMA;

[0009] Step 4. Use a real-time fluorescence quantitative nucleic acid amplification detection system qPCR to detect the expression of macrophage marker CD11b. If the expression of CD11b is positive, it is identified as a macrophage.

[0010] The beneficial effect of the present invention is that the macrophages cultured by this method have a higher purity and a faster growth cycle, which is conducive to experimental research. Brief Description of the Drawings

[0011] To more clearly illustrate the technical solutions in the embodiments of the present invention or in the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0012] Figure 1 It is a flowchart of the macrophage culture method provided by the embodiment of the present invention. Specific embodiments

[0013] The following will clearly and completely describe the technical solutions of the present invention in combination with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0014] Example 1

[0015] The THP-1 cells were isolated and established from the peripheral blood of a 1-year-old boy with acute monocytic leukemia. The cells can phagocytize latex particles and activated red blood cells, and there are no immunoglobulins in the cell membrane and cytoplasm, and they express C3R and FcR. The THP-1 cells can be induced to differentiate in the monocytic direction by phorbol ester and TPA. The THP-1 cells can be used for 3D cell culture, immune system disorders, immunology and toxicology research, and are also a suitable transfection host.

[0016] Experimental materials

[0017] 1. Main reagents

[0018] Phorbol ester (PMA) Sigma-Aldrich

[0019] RPMI-1640 Sevier Bio

[0020] 2. Main instruments

[0021] CO2 incubator Thermo Fisher 3111

[0022] As Figure 1 shown, the macrophage culture method includes the following steps:

[0023] Step 1: Suspend the THP-1 cells in a T25 culture flask with 6-8 mL of RPMI-1640 complete medium and place it in a 37°C / 5% CO2 incubator for culture. Add 1-2 mL of fresh medium to the flask every 2-3 days. When the cell density reaches 8x105-x106 cells / mL, passage the cells;

[0024] Step 2. When the cells grow to an appropriate concentration, add phorbol 12-myristate 13-acetate (PMA) to the culture flask to a final concentration of 100 ng / mL. Gently mix and then place it in a CO2 incubator for culture;

[0025] Step 3. Observe the cell adhesion after stimulation with phorbol 12-myristate 13-acetate (PMA) for 24 h;

[0026] Step 4. Use a real-time fluorescence quantitative nucleic acid amplification detection system (qPCR) to detect the expression of macrophage marker CD11b. If the expression of CD11b is positive, it is identified as a macrophage.

[0027] Step 5. Use THP-1-derived macrophages for subsequent experiments.

[0028] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. A method for culturing macrophages, characterized in that, Including the following steps: Step 1: Suspend human monocytic leukemia THP-1 cells in a T25 culture flask with 6-8 mL of complete RPMI-1640 medium, and place it in a 37°C / 5% CO2 incubator for culture. Add 1-2 mL of fresh medium to the flask every 2-3 days. Passage the cells when the cell density reaches 8x105 - x106 cells / mL; Step 2. When the cells grow to an appropriate concentration, add phorbol 12-myristate 13-acetate (PMA) to the culture flask to a final concentration of 100 ng / mL. Gently mix and then place it in a CO2 incubator for culture; Step 3. Observe the cell adhesion after 24 hours of stimulation with phorbol 12-myristate 13-acetate (PMA); Step 4. Use a real-time fluorescence quantitative nucleic acid amplification detection system qPCR to detect the expression of macrophage marker CD11b. If the expression of CD11b is positive, it is identified as a macrophage.

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