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Macrophages are critical in the body's immune system, playing an important role in phagocytizing pathogens, regulating immune responses, and maintaining tissue homeostasis. Through the primary extraction method, researchers can avoid the genetic changes and functional decline that may be caused by multiple passages of cell lines, and directly obtain macrophages with complete biological activity from living organisms which lays a solid foundation for subsequent experiments.

Primary bone marrow-derived macrophages are usually round or oval when unstimulated, with varying cell sizes, small nuclei, mostly round or oval, loose chromatin, and obvious nucleoli. Vacuoles and granular substances are common in the cytoplasm, and these structures are closely related to their phagocytic function.

Centrifuges play a vital role in the BMDM cell extraction process. Through the centrifugation step, we can effectively separate and precipitate cells to obtain high-quality bone marrow macrophages. The centrifugation process not only helps remove impurities, but also ensures the purity and activity of cells, which is an indispensable key link in the extraction process.

The following are the detailed steps and precautions for the primary macrophage extraction method using mice as an example:

Experimental material preparation

Experimental animals: C57BL/J mice

Reagents: DMEM high-glucose culture medium containing 10% FBS, 75% ethanol, sterile PBS.

Experimental equipment: disposable syringes, surgical instruments, centrifuge tubes, centrifuges, cell culture plates or culture flasks, etc.

Experimental steps

●Anesthesia and killing: After anesthetizing the mice, they were killed quickly and humanely by dislocation, following the ethics of animal experiments and minimizing the pain of the mice.

●Surface disinfection: Soak the mice in a beaker containing 75% ethanol for 5 minutes. After thorough disinfection, use a clean paper towel to absorb the excess alcohol.

●Skin and joint treatment: Use sterilized scissors to cut a small incision on the back of the mouse, tear the skin to the calf joint with bare hands, and remove the foot joint and skin.

●Leg sampling and secondary disinfection: Use sterilized scissors to carefully disassemble the hind limbs from the greater trochanter at the root of the thigh to avoid fractures. After removing the muscles, soak the leg bones in a 75% ethanol culture dish for 5 minutes.

●After 5 minutes, take the leg bones out of the ethanol culture dish, place them in a new ethanol culture dish, and move them to the clean bench to provide a sterile environment for subsequent experiments.

BMDM cell extraction and induction

● Leg bone pretreatment: Transfer the ethanol-soaked mouse leg bones to a container filled with cold PBS, completely immerse and rinse the ethanol on the bone surface with PBS, repeat 3 times to ensure thorough cleaning.

● Bone marrow extraction: Separate the cleaned femur and tibia, and cut both ends with sterilized scissors. Use a 1mL syringe to absorb cold induction medium, aim at the bone end and blow out the bone marrow, and blow and wash repeatedly for about 3 times until there is no obvious red bone marrow residue in the leg bone.

● Cell dispersion and filtration: Use a 5mL pipette to gently blow the medium containing bone marrow cells to disperse the cell clumps. Filter the suspension through a 70μm cell filter to remove impurities, and transfer the filtrate to a 15mL centrifuge tube.

● Centrifugation and resuspension: Centrifuge at 1500rpm for 5 minutes and discard the supernatant. Add red blood cell lysis buffer to resuspend the cells, let it stand for 5 minutes, and then centrifuge at the same speed for 5 minutes, and discard the supernatant. Finally, resuspend the cell pellet with cold induction medium.

● Cell culture and medium replacement: Plate the cells as required. On the third day, half of the culture medium was replaced to maintain a stable culture environment; on the fifth day, a full amount of fresh culture medium was replaced to meet the needs of cell growth and differentiation; on the seventh day, the cells reached a usable state.

Notes

●BMDM cells cannot be passaged: Due to the special biological characteristics of BMDM cells, they are not suitable for passage operations.

●Avoid trypsin digestion: Trypsin is often used to separate adherent cells, but it is not suitable for BMDM cells. The proteolytic activity of trypsin will destroy the cell membrane and key proteins, causing cell inactivation and unusable for subsequent experiments. Therefore, do not use trypsin to treat BMDM cells.

●It is recommended to use a cell scraper: Due to the limitations of passage and trypsin digestion, it is recommended to use a cell scraper to gently scrape BMDM cells.

●Avoid frequent changes of culture containers: BMDM cells are fragile and sensitive to the environment. It is recommended to directly plate them for experiments after acquisition. Frequent changes of culture containers may cause physical disturbances, causing cell collisions and pulling, thereby damaging cells and interfering with experimental results.

The above is the detailed process of BMDM cell extraction which will be helpful to the users. In biological experiments, Welso can provide you with all the experimental instruments you need. It is particularly worth mentioning that our centrifuge series is an indispensable assistant in the experiment. We focus on providing high-quality centrifuge products to global customers, including low-speed centrifuges, high-speed centrifuges, refrigerated centrifuges, mini centrifuges, and large-capacity floor centrifuges. If you are interested in our products, please contact us in time and you will get the best quotation.