Macrophages (MΦ) are mature cells formed by the migration of blood monocytes into tissues, and are widely distributed in the spleen, liver, abdominal cavity, nervous system, and connective tissues, with phagocytosis and killing, antigen presentation, and immunomodulatory effects. Recent studies have found that macrophages can be activated in different microenvironments and become subpopulations with different surface molecular and functional characteristics. According to their different phenotypic and functional characteristics and induction of Th1/Th2 responses, they are usually classified into two types: classically activated macrophages (CAM), also known as M1-type macrophages; alternatively activated macrophages (AAM), also known as M1-type macrophages; and alternatively activated macrophages (AAM), also known as M1-type macrophages. M1-type macrophages have microbicidal, inflammatory and antitumor effects, while M2-type macrophages have strong immunomodulatory, tissue repair and anti-inflammatory abilities. In recent years, it has been found that tumor-associated macrophages (TAM) in the tumor mesenchyme have the characteristics of the M2 type, and TAM are involved in tumorigenesis, growth, invasion and metastasis through the secretion of EGF, VEGF and bFGF, and are closely related to tumor angiogenesis and lymphangiogenesis; TAM are also closely related to tumor angiogenesis and lymphangiogenesis; TAM are also closely related to tumor angiogenesis and lymphangiogenesis. TAM is involved in tumorigenesis, growth, invasion and metastasis through the secretion of EGF, VEGF and basic fibroblast growth factor (bFGF), and is also closely related to tumor angiogenesis and lymphangiogenesis.
Principle
The basic principle of isolation and purification of mouse peritoneal macrophages is that macrophages are nonpropagating cells, which can survive for 2-3 weeks under suitable conditions, but are difficult to survive for a long period of time, and are mostly cultured in primary cultures. MΦ can be cultured from different sources by various methods, among which the mouse peritoneal sampling method is the most commonly used to isolate and purify MΦ from mouse peritoneal exudate cells, and about 106 peritoneal exudate cells can be collected from each normal mouse, of which MΦ accounts for 30%. If a certain stimulus is injected into the peritoneal cavity of mice, the exudate of MΦ can be increased and more MΦ can be easily isolated.
Operation method
Isolation and purification of mouse peritoneal macrophages by applanation method
Principle
Macrophages are nonpropagating cells that can survive for 2-3 weeks under appropriate conditions, but are difficult to survive for long periods of time and are mostly cultured in primary cultures. Various methods can be used to culture MΦ from different sources, among which the peritoneal sampling method is the most commonly used, to isolate and purify MΦ from mouse peritoneal exudate cells, and normal mice can collect about 106 peritoneal exudate cells per mouse, of which MΦ accounts for 30%. The basic principle of wall sticking method is based on the characteristic of monocyte macrophage adhering to plastic or glass surface, which can separate monocyte macrophage and lymphocyte.
Materials and Instruments
Animals: 6~8 weeks old C57/BL6J mice. Move (1) The cervical vertebrae of mice were dissected and killed, sterilized with 75% ethanol, and transferred to an ultra-clean table and fixed on an anatomical fixation plate in supine position. (2) Cut open the abdominal fur layer to expose a large area of peritoneum, avoiding blood vessels. 5 ml of RPMI 1640 culture medium containing double antibody should be injected into the peritoneal cavity, and the abdomen should be gently rubbed for 1~2 minutes, and then left to stand for 5 minutes. 3）Use forceps to lift the peritoneum to cut a small opening (the pipette tip can be entered), use a pipette to suck up the fluid in the peritoneal cavity (pay attention to avoid the intestinal tube), and put it into a centrifuge tube. (4) Centrifuge the cells at 1,500 r/min for 5 minutes, then wash them once with RPMI 1640 medium containing double antibody, then centrifuge the cells, add fresh culture medium to resuspend the cells and count them. (5) Adjust the cells to the desired concentration, place them on a flat dish or cell culture plate, and incubate at 37 ℃ in CO2 incubator for 1~2 hours. 6) Remove the culture medium, wash the cells with pre-warmed culture medium at 37 ℃ for 2 times, discard the unadhered cells, the adherent cells are monolayer macrophages, and then add appropriate culture medium to culture. Caveat (1) Inject the lavage fluid into the needle from the side of the lower abdomen to ensure that the eye of the needle is constricted and does not bleed from the eye of the needle as a result of the increase in peritoneal fluid and to avoid mixing of blood cells into the macrophage suspension.(2) The abdomen should be gently rubbed after the intraperitoneal injection, both to avoid blood seepage and to ensure that the macrophages are free, and then the lavage should be performed thoroughly to obtain as many macrophages as possible. If the aspirated lavage fluid is yellowish, more macrophages are obtained; if the aspirated lavage fluid remains colorless and transparent, fewer macrophages are obtained. Analyze this phenomenon may be free from a large number of macrophages to make its environment become acidic.(3) Opening the abdominal cavity under aseptic conditions and aspirating under direct vision prevents the phenomenon of blocking the needle eye due to aspiration of mesentery or peritoneum, etc., and allows rapid and more complete aspiration of lavage fluid.(4) When collecting inflammatory peritoneal macrophages, inject 1 ml of 3% guinea pig peptone broth or 2 ml of sterile liquid paraffin into the peritoneal cavity of mice with a syringe before executing the mice, and then collect them according to the above steps after 3~4 days. For more product details, please visit Aladdin Scientific website.
Reagents: RPMI 1640 medium (containing 10% fetal bovine serum, double antibody), PBS liquid, 75% alcohol.
Instruments: anatomical fixation plates, test tubes (or centrifuge tubes), syringes, scissors, tweezers, cell culture plates (or petri dishes), cell counting plates, pipettes and tips, ultra-clean table, carbon dioxide incubator, centrifuge.