Introduction of a method for the isolation of mouse macrophages by J.E. Colligan et al, translated by Xuitao Cao et al. This experiment is from the "Compendium Immunology Laboratory Guide".
Operation method
Isolation of mouse macrophages Move Basic protocol 1 Isolation of mouse peritoneal macrophages Materials Mice, housed in a clean environment (preferably in a laminar flow cabinet) 7 3 % (m/V) guinea pig peptone or 3 % (WVr) Brewer thioacetate broth (Brewerthioglycollate medium for isolation of inflammatory abdominal macrophages) 70 % ethanol Collection medium: D M E M (G I B C O /B R L ) with 5 % (v/V ) FBS (Hyclone, endotoxin free). Diff-Quik stains I, II and III (Baxter) 25 G Needle 6m l and 30m l syringes Tweezers and flathead surgical scissors (immersed in 70% ethanol) 19 G needles 50 m l polypropylene cone-bottomed plastic centrifuge tube, pre-chilled on ice Centrifuge (Shandon/Lipshaw) Cell Flaker (Shandon/Lipshaw) Sorvall R T 6000 centrifuge and H l O O O B rotor (or alternative) la. For collection of resting peritoneal macrophages: decapitation or CO2 asphyxiation of mice (Appendix 2G). Ib. For collection of inflammatory peritoneal macrophages: Draw up Iml 3 % guinea pig broth in a 6 ml syringe. The broth is injected into the peritoneal cavity of the mouse using a 25 G needle (Appendix 2E). Mice are killed by decapitation or CO2 asphyxiation after 3d (Appendix 2G). Alternatively, Iml 3 % Brewer sulfoacetate broth can be injected intraperitoneally and the cells collected after 5-7d. 2.70% ethanol is used to sterilize the mouse abdomen. The abdominal fur was aseptically clipped and held with forceps and torn to both sides to expose the intact peritoneum. 3 . Attach a 19 G needle to a 30 ml syringe and aspirate IO ml of collection medium. The tip of the needle is beveled upward, piercing the peritoneum at the midline (Appendix 2E), and the medium is injected into the peritoneal cavity. To prevent puncturing the intestinal wall, push the syringe so that a small amount of medium flows through the needle and into the peritoneal cavity as the needle tip passes through the peritoneum. 4 . Adjust the tip of the needle with the beveled side down, gently pick up the peritoneum, and slowly aspirate back the abdominal lavage fluid (about 8 ml per mouse). Add the peritoneal lavage fluid to a 50 ml polypropylene centrifuge tube pre-cooled on ice. 5 . Take 20 ul of Peritoneal lavage fluid and count (Appendix 3A). 6 . Add 0.2 ml of ascitic lavage fluid to the cell flaker. Centrifuge at 600r/m i n for 6 min as instructed and prepare the cell slides. After air-drying, the cells were stained with Diff-Quik and counted. 7 . Centrifuge the remaining peritoneal lavage fluid at 400 g for lOmin at 4°C. Discard the supernatant and flick the bottom of the tube to homogenize the cells. Add collection medium to adjust the cells to the appropriate concentration. A mouse can obtain 2 X 106 to 3 X 106 peritoneal cells, of which 50% to 70% are macrophages. In mice injected intraperitoneally with peptone or thioglycolate broth, 3 X IO6 to 4 X IO6 and about IO7 macrophages were obtained per mouse, respectively. These proinflammatory agents recruit new, immature macrophages into the abdominal cavity. Mice, kept in a clean environment (preferably in a laminar flow cabinet) PBS Lymphocyte Separation Solution (LSM; Organon Teknika Cappel) E M E M and E M E M -10 with additives Mouse-specific CSF or IL-3 (GENZYME) Neutrophil type II, 1.0m g / m l (BoehringerMannheim) Tweezers and scissors (immersed in 70 % ethanol) 25m l syringe 26 G Needle 50 m l polypropylene cone-bottomed plastic centrifuge tube, pre-chilled on ice 15 m l polypropylene cone-bottomed plastic centrifuge tube Sorvall R T 6 0 0 0 centrifuge and H B 1 0 0 0B rotor (or alternative) 25c m 2 and 75c m 2 cell culture bottles (Corning) Sterile rubber cell scraper 1 . Execute the mice (Appendix 2 G ). Strip the fur from the hind limbs to the feet. Clip the feet along with the removed fur. Cut off the hind legs and place them in a petri dish containing sterile PBS. 2 . After holding the leg bone at one end with forceps, remove the muscle with scissors. Cut the leg bone at the joint For more product details, please visit Aladdin Scientific website.