Mouse peritoneal macrophage culture experiment
Mouse peritoneal macrophage culture experiment
Mouse peritoneal macrophage cultivation can (1) phagocytosis and removal of foreign substances; (2) secretion of bioactive substances; (3) to bionic fully degradable materials, the role of degraded inorganic products and the synthesis of organic substances in the life process.
Operation method
Cell Culture Technology
Principle
Mouse peritoneal macrophages were taken and mixed with latex particles under different culture conditions and then quantitatively added into 24-well plates, and the phagocytic percentage and phagocytic index were counted under a fluorescence microscope after incubation at 37 ℃ in a 5% CO2 incubator.
Materials and Instruments
Mouse Move I. Experimental steps Caveat 1. strict aseptic operation in an ultra-clean table;2. To avoid cross-infection, change the syringe for each mouse;3. when aspirating the cell suspension by pipette, try not to aspirate to the large and small intestine, otherwise it will easily cause contamination of fibroblasts. Common Problems Macrophages are generally not specifically identified by two lessons: 1. macrophages are terminally differentiated cells and do not proliferate 2. they are difficult to digest, so when inoculated, they must be inoculated directly into the intended culture vessel. For more product details, please visit Aladdin Scientific website.
1640 culture medium calf serum double antibody Taipanlan
Surgical Instruments Disposable Syringes Ophthalmic Forceps Ophthalmic Scissors 96-well Plates CO2 Incubator
1. If stimulants are used, a higher number of macrophages can be obtained by intraperitoneal injection of 3% sodium thioglycolate 2 mL each three days before the experiment. Without using stimulants, start the following steps directly.2. Pull the neck to execute the mice, soak them in 75% alcohol for 1-2 minutes, transfer them into an ultra-clean table, fix them on a dissecting board in the supine position, sterilize the abdominal skin with iodine, and deiodinate them with alcohol wool balls. The abdominal skin was cut open with straight surgical scissors to expose the abdominal musculature, which was then sterilized with alcohol pellets.3. 5mL of RPMI 1640 medium containing double antibody was injected into the abdominal cavity with a syringe, and the abdomen was gently rubbed with a cotton ball for 1-2 min, then the abdominal cavity was slightly lifted up with ophthalmologic forceps and a small opening was cut with ophthalmic scissors, and the cell suspension was sucked up with a pipette and transferred into a centrifuge tube (I personally think that this method is better than using a syringe to suck up the cell suspension).4. After centrifugation at 1000 rpm for 5 min, the cells were washed once with RPMI 1640 medium containing double antibody, centrifuged again, and resuspended in RPMI 1640 medium containing 10% calf serum, or in the case of feeder cells, the corresponding medium. The cells were counted by the Taipan blue rejection method, diluted to the target concentration and then inoculated.5. If used as feeder cells, adjust the concentration to 2x105 cells/mL, inoculate into 96-well plate, 100-200μL per well; if used for other experiments, adjust to 2x106/mL, add into 24-well flat-bottomed plate, 1 mL/well; incubate in 5% CO2 oven for 2 h, then change the liquid, and wash with RPMI 1640 medium for 1-2 times, and discard. After incubation for 2 h in 5% CO2 incubator, change the solution and wash with RPMI 1640 medium for 1-2 times, discard the unadhered cells, and the adherent cells were monolayer macrophages.
Results
Macrophages were rounded or pebble-like when they first adhered to the wall, and then slowly extended their pseudopods and spread out in a triangular or polygonal shape. Macrophages have the property of phagocytosis, when mixed with bacteria, macrophages can be seen phagocytosis of bacteria under the 40x objective, therefore, macrophages as feeder cells can remove part of the contaminated bacteria, mycoplasma, and part of the cell debris.