Mouse peritoneal macrophage culture can: (1) phagocytosis and removal of foreign bodies; (2) secretion of biologically active substances; (3) synthesis of biomimetic fully degradable materials, degraded inorganic products and organic matter in life processes. Xmas Gummy Candy,Christmas Gummies,Christmas Gummy Candy,Fruity Christmas Candy Montreal Shantou Food Co., Ltd , https://www.montrealsnack.com experimental method
Principle of experimental method Mouse peritoneal macrophages and latex particles were mixed under different culture conditions, and then quantified into 24-well plates. After incubation at 37 ° C in a 5% CO 2 incubator, the percentage of phagocytosis and phagocytic index were counted under a fluorescence microscope. Experimental Materials Reagents, kits Instruments, consumables Experimental procedure
Second, the results Precautions other
Macrophages generally have no special identification methods and have two experiences:
1. Macrophages are terminally differentiated cells that do not proliferate
2. It is difficult to digest, so when inoculation, it must be directly inoculated into the target culture vessel.
Cell Technology Topic: Mouse Peritoneal Macrophage Culture Experiment
Mouse
1640 medium calf serum double-resistant trypan blue
Surgical instruments, disposable syringes, ophthalmology, ophthalmology scissors, 96-well plate, CO2 incubator
First, the experimental steps
1. If irritant is used, intraperitoneal injection of 3% sodium thioglycolate 2 mL each 3 days before the experiment will result in more macrophages. Instead of using irritants, start the following steps directly.
2. The mice were sacrificed by pulling the neck, soaked in 75% alcohol for 1-2 minutes, transferred to the ultra-clean table, the supine position was fixed on the anatomical plate, the iodine disinfected the abdominal skin, and the alcoholic cotton ball was deiodinated. The abdominal skin is fully cut with a surgical straight cut, the abdominal muscle layer is exposed, and the abdominal muscle layer is disinfected with an alcohol sponge.
3. Using a syringe, inject 5 mL of RPMI 1640 medium containing double antibody into the abdominal cavity, and gently rub the abdomen with a cotton ball for 1-2 min. Then, use the ophthalmology to lift the abdominal cavity slightly, and open a small mouth with an ophthalmology scissors to absorb the cell suspension with a pipette. The liquid is transferred into a centrifuge tube (personally believes that this method is better than using a syringe).
4. After centrifugation at 1000 rpm for 5 min, wash once with RPMI 1640 medium containing double antibody, centrifuge again, and resuspend the cells in RPMI 1640 medium containing 10% calf serum. If used as feeder cells, select The corresponding culture solution. The trypan blue exclusion method is counted, and the cells are diluted to the target concentration, and then inoculated.
5. If used as a feeder, adjust the concentration to 2x10 5 /mL, inoculate into a 96-well plate, 100-200 μL per well; if used as other experiments, you can adjust to 2x10 6 /mL, add to 24 holes In a flat-bottomed culture plate, 1 mL/well; incubated in a 5% CO 2 incubator for 2 h, change the solution, and wash 1-2 times with RPMI 1640 medium, discard the non-adherent cells, and attach the cells to a single layer. Phagocytes.
The macrophage is rounded when it is attached to the wall, or resembles a cobblestone shape, and then slowly protrudes from the pseudopod and spreads out in a triangular or polygonal shape. Macrophages have the characteristic of phagocytosis. When mixed with bacteria, macrophages can be seen to phagocytose bacteria under 40 times objective lens. Therefore, macrophages can be used as feeder cells to remove some contaminated bacteria, mycoplasma and some cells. Fragmentation.
Precautions: Strict aseptic operation, in a clean bench; to avoid cross-infection, each mouse is replaced with a syringe; when sucking the cell suspension, try not to suck the large and small intestine, otherwise it may cause fibroblast contamination. .
Expand  I. Discussion