Primary culture of mouse abdominal macrophages can be applied to (1) cell preservation; (2) molecular biology research; (3) gene therapy research.
Operation method
laparotomy (medicine)
Principle
Macrophages are immune cells with a variety of functions, and are important targets for studies of cell phagocytosis, cellular immunity and molecular immunology. Macrophages are easy to obtain, easy to culture, and can be purified. Macrophages are nonpropagating cells that can live for 2-3 weeks under suitable conditions, and are mostly used for primary culture, making long-term survival difficult.
Materials and Instruments
Mouse Move I. Preparation of experimental materials Caveat 1. Macrophages are terminally differentiated cells and do not proliferate. 2. It is difficult to digest down, so when inoculating, it must be directly inoculated into the target culture vessel. 3. Strictly aseptic operation is carried out in the ultra-clean table. 4. To avoid cross-infection, change the syringe for each mouse. 5. when sucking cell suspension by pipette, try not to suck into the large and small intestines, otherwise it is easy to cause contamination of fibroblasts. Common Problems Macrophages are rounded or pebble-like when first attached to the wall, then slowly extend pseudopods and spread out in a triangular or polygonal shape. Macrophages have phagocytic properties and when mixed with bacteria, macrophages can be seen phagocytizing bacteria under a 40x objective lens; therefore, macrophages act as feeder cells to remove some of the contaminating bacteria, mycoplasmas, and some of the cellular debris. For more product details, please visit Aladdin Scientific website.
RPMI1640 medium Phenol red Alcohol Calf serum Sodium mercaptoethanolate Dual antibody Culture medium Calf serum Penicillin Streptomycin Teparan Iodine
Sheep ball Surgical straight scissors Syringe Petri dish Ultra-clean table Anatomical plate Ophthalmic scissors Centrifuge tube Ophthalmic forceps
1. Animals: 2-4 mice of each strain.
2. Reagents: RPMI1640 medium (containing phenol red), calf serum, double antibody, culture medium (10% calf serum, RPMI1640, double antibody), Taipan orchid and so on. Iodine wool balls and alcohol wool balls.
3. Instruments: disposable syringes, surgical instruments.
Experimental methods
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, the following steps were started directly.
1. Pull the neck to execute the mice, soak them in 75% alcohol for 1-2 min, 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 fully cut with surgical straight scissors to expose the abdominal musculature, and then the abdominal musculature was sterilized with alcohol wool balls.
2. 5 mL of RPMI1640 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 ophthalmologic 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).
3. After centrifugation at 1,000 rpm for 5 min, the cells were washed once with RPMI1640 medium containing double antibody, centrifuged again, and resuspended in RPMI1640 medium containing 10% calf serum, or if used as feeder cells, the corresponding medium was selected. The cells were counted by Taipan blue rejection staining method, diluted to the target concentration and then inoculated.
4. If it is used as feeder cells, adjust the concentration to 2x105/mL, inoculate into 96-well plate, 100-200 uL per well; if it is used as other experiments, it can be adjusted to 2x106/mL, add into 24-well flat-bottomed culture plate, 1 mL/well; after incubation for 2 h in 5% CO2 incubator, change the liquid, and wash with RPMI1640 culture medium for 1-2 times, discard the unadhered cells, and the adherent cells were macrophages in monolayer.
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 40x objective, therefore, macrophages as feeder cells can remove some of the contaminated bacteria, mycoplasma and some cell debris.