Induction and characterization of macrophage polarization in mice
Induction and characterization of macrophage polarization in mice
Macrophages can undergo functional activation (polarization) of different nature into M1 and M2 subpopulations with different surface molecular and functional characteristics.M1-type macrophages are effector macrophages generated during Th1-type immune responses, mainly induced by microbial products (e.g., LPS) or interferon gamma (IFN-Y). They are characterized by the secretion of killer molecules such as nitric oxide (NO), reactive oxygen intermediates, and a variety of inflammatory factors (IL-1, IL-6, IL-12, TNF-α, and Type IIFN, etc.) and chemokines (CXCL8, CXCL9, CXCL10, CXCL11, CXCL16, CCL2, CCL3, CCL4, and CCL5, etc.). In addition, under the stimulation of IFN-Y, macrophages can increase the secretion of inducible nitric oxide synthase (iNOS), and express a large number of molecules such as CD16/32, MHC II, CD40, CD80, CD86, and B7, which are involved in the presentation of antigens and the stimulation of the Thl-type immune response, killing infectious agents and tumor cells. The M1 macrophages are also able to express a large number of molecules such as CD16/32, MHC II, CD40, CD80, CD86, and B7, which are involved in antigen presentation and stimulation of the Thl-type immune response. Therefore, it is generally believed that M1-type macrophages are powerful effector cells for immune defense and antitumor activity. M2-type macrophages differentiate into different subtypes in response to different inducing signals, including: ① M2a-type, which stimulates IL-4 and IL-13, induces Th2-type immune responses, and plays a major role in allergic and parasitic infections. Type M2b, in which the stimulatory signals are immune complexes and Toll-like ligands (e.g., LPS) or IL-1R ligands, mediate Th2-type activation and immunoregulation. The characteristic phenotypes of M2 macrophages are IL-10 hih, IL-12 low, IL-1rahih, IL-1decoyRhigh, which can secrete chemokines (e.g., CCL17, CCL18, CCL22) and specifically express some genes, such as type 1 genes. It also expresses genes such as the gene encoding arginasel type 1 (Arg1), and highly expresses mannose receptor CD206, scavenger receptor, and up-regulates the expression of secretory proteins such as YM1 and YM2 (members of the chitinase family), FIZZ1, etc. The M2-type macrophages are poor in antigen presentation and control the inflammatory response, including through the down-regulation of the function of M1-type macrophages. M2-type macrophages are poor in antigen presentation and control the inflammatory response in the body by down-regulating the function of M1-type macrophages. At the same time, M2-type macrophages have the ability to repair damage, promote angiogenesis and tissue remodeling.
Principle
The basic principle of the induction of macrophage polarization in mice is to induce macrophage polarization by using the appropriate inducers; the phenotypes of macrophage types can be evaluated and identified through the comparative analysis of the indicators related to the phenotypes of M1 and M2 types of macrophages, such as the markers of the M1 type (CD16/32, IL-1β, IL-6, TNF-α, IL-12, NO and iNOS, etc.), the markers of the M2 type (CD206/mrc-1, IL-10, Arg-1 and Yml, etc.), and the markers of the M2 type (CD206/mrc-1, IL-10, Arg-1, and Yml, etc.). M1-type markers (CD16/32, IL-1β, IL-6, TNF-α, IL-12, NO and iNOS, etc.) and M2-type markers (CD206/mrc-1, IL-10, Arg-1 and Yml, etc.).
Operation method
Induction and characterization of macrophage polarization in mice
Principle
Macrophage polarization was induced using the appropriate inducers, respectively; the phenotypes of macrophage types could be evaluated and identified by comparative analysis of the indicators related to the phenotypes of M1 and M2 types of macrophages, such as the markers of the M1 type (CD16/32, IL-1β, IL-6, TNF-α, IL-12, NO and iNOS, etc.), and the markers of the M2 type (CD206/mrc-1, IL-10, Arg-1 and Yml, etc.).
Materials and Instruments
Animals: Move I. Induction of macrophage polarization 1) Isolate mouse peritoneal macrophages, adjust the cell concentration to 1 × 106/mL, and add to 12-well or 24-well plates. (2) Induction of M1 type MΦ polarization: LPS (1 μg/mL, final concentration) and IFN-y (20 ng/mL, final concentration) were added to the culture medium and cultured for 24-96 hours. (3) Polarization induction of M2a type MΦ: IL-4 (20 ng/mL, final concentration) was added to the culture medium and incubated for 24-96 hours. (4) Polarization induction of M2b type MΦ: Immunocomplex-anti-LDL (LDL 100 μg/mL, final concentration) and LDL (5-10 μg/mL, final concentration) or human IgG (10 μg/mL, final concentration) and rabbit anti-human IgG (10 μg/mL) were incubated for 24~96 hours. (5) Polarization induction of M2c-type MΦ: IL-10 (20 ng/mL, final concentration) was added to the culture medium and cultured for 24~96 hours. (6) Morphological observation of polarized macrophages: the morphology of macrophages changed after polarization; macrophages polarized toward M1 type showed a long shuttle shape with elongated pseudopods; the pseudopods of M2 type polarized macrophages were shorter, and the overall morphology of the cells was more convergent. (7) Identify and analyze the types of macrophage polarization. Identification of macrophage polarization type Different indicators can be detected by different technical means: collect the above cells and cell culture supernatants, detect the expression of cell membrane molecules CD16/32 and CD206 by flow cytometry, analyze the expression and secretion of the characteristic factors IL-1β, IL-6, TNF-α, IL-12 and IL-10 by real-time quantitative PCR and ELISA kit, and analyze the expression and secretion of iodine by real-time quantitative PCR or Western blot. The expression and secretion of the characteristic factors IL-1β, IL-6, TNF-α, IL-12 and IL-10 were analyzed by real-time quantitative PCR or Western blot. The following is a list of several ways to identify macrophage polarization types, which can be combined according to the situation, and at least 4 indexes for each polarization type are selected as typing characteristics. (1) Macrophage NO secretion: Collect the supernatant of macrophages treated as described above, and use Griess Reagent to detect NO, as described in the Griess Reagent instructions. (2) Detection of macrophage surface markers by flow cytometry: Macrophages after induced polarization were collected by cell scraping, and identified by flow cytometry using fluorescent labeled antibodies such as anti-CD16/32, anti-iNOS, and anti-CD206. (3) real-time PCR to detect the expression of macrophage surface markers and cytokine genes: total RNA of macrophages was extracted and reverse transcribed to obtain cDNA, and primers were designed according to the gene sequences of mouse IL-1β, IL-6, TNF-α, IL-12, IL-10, iNOS, Arg-1, and YmL recorded in the Genbank, and then analyzed the macrophages by real-time quantitative PCR. Real-time quantitative PCR was used to quantify the surface markers and cytokines of macrophages at the transcription level. (4) Western blot analysis of the expression of surface markers and cytokines in macrophages: total proteins of macrophages were extracted, and the expression of iNOS, Arg-1, and YmL was detected by Western blot analysis. (5) Detection of cytokines and chemokines secreted by macrophages by ELISA kit: Collect the supernatants of macrophages treated above, and use ELISA kit to detect the secretion of cytokines IL-1β, IL-6, TNF-α, IL-12, and IL-10; and use ELISA kit to detect the secretion of chemokines CCL2, CCL3, and CCL22, etc. For the specific procedures, see ELISA kit, and see ELISA kit, and use ELISA kit to detect the secretion of CCL2, CCL3, and CCL22. For details, please refer to the instructions of ELISA kit. (6) Macrophage phagocytosis in vitro by fluorescent microsphere assay: Incubate treated red fluorescent phagocytosis particles with mouse peritoneal macrophages at 37 ℃ for 1 hour, and detect the phagocytosis of fluorescent microspheres by fluorescent microscope, or detect the phagocytosis percentage by flow cytometry. (7) Measurement of Arg-1 activity: Macrophages were lysed with 100 μl of 0.1% TritonX-100, 100 μl of 50 mmol/L Tris-HCl and 10 mmol/L MnCl2 were added, and incubated at 56 ℃ for 10 minutes. 100 μl of 0.5 mol/L Arginine was added, and incubated at 37 ℃ for 30 minutes, and terminated by adding 800 μl of H2SO4. The reaction was terminated by adding 800 μl of H2SO4. Then add 50 μl of 9% α-isonitrosopropiophenone and incubate at 95 ℃ for 30 min. The absorbance (A value) was detected at 540 nm, and a standard curve was set up with urea to indirectly determine the activity of Arg-1. Caveat (1) Observe the cells with naked eyes and microscope for contamination during the culture process.(2) If the culture supernatant is used to test the relevant indexes, the supernatant should be centrifuged before testing, so as to avoid mixing with broken cells to affect the experimental results. For more product details, please visit Aladdin Scientific website.
6-8 weeks old C57/BL6J mice
Reagents:
1) LPS, IFN-Y, IL-4;
2) Cytokine and chemokine detection kits (e.g. IL-1β, IL-6, TNF-α, IL-12 and IL-10, etc.; CXCL8, CXCL9, CXCL10, CCL17, CCL18 and CCL22, etc.);
(3) real-time PCR reagents, iNOS, Arg-1, YmL, Fizz1 and various cytokine or chemokine primers;
(4) Griess reagent kit for NO detection;
(5) Flow cytometry and Western blot detection reagents, anti-CD16/32, anti-iNOS, anti-CD206 and other labeled antibodies, PVDF membrane;
6) Macrophage phagocytosis function detection: red fluorescent phagocytosis microspheres.
Instruments:
1) Enzyme labeling instrument;
(2) real-time PCR instrument;
(3) Flow cytometer;
(4) Vertical electrophoresis instrument, transfer electrophoresis tanks