BMDM培养

BMDM的培养Isolation of Bone Marrow-derived Macrophages2010.2.24-CCFDevelopment of the macrophage lineage of cells from bone marrow progenitors is regulated by macrophage colony-stimulating factor (M-CSF), which is constitutively produced by many cells types. In this protocol, bone marrow cells are grown in culture dishes in the presence of M-CSF, which is secreted by L929 cells and is used in the form of L929-conditioned medium. Under these conditions, the bone marrow monocyte/macrophage progenitors will proliferate and differentiate into a homogenous population of mature BMMs.L929 medium:DMEM (high glucose)10% fetal bovine serum penicillin-streptomycin-glutamine Preparation of L929-conditioned medium: 1. Plate L929 cells in a 15-cm2 plates containing 25 mL of L929 medium. 2. Grow cells in a humidified incubator with 5% CO2 at 37C , change medium the next day.3. Grow cells in a humidified incubator with 5% CO2 at 37C , collect the supernatant the next day, save and store at -20C. Add more media into the plates.4. Repeat step 3 two more times.Preparation of bone marrow growth medium:DMEM 50%fetal calf serum 20%L929-Conditioned Medium 30%penicillin-streptomycin-glutamine 1. 第0天：清洗2套镊子，剪刀，在hood中紫外照射半小时。2. 颈脱臼处死小鼠，在75%的酒精中浸泡几分钟。3. 在泡沫板上用针头固定小鼠上肢，然后用剪刀剪开小鼠脚趾处皮肤，继续向上剪至腹部，剥离出小鼠后肢。4. 沿肌肉走向剪开肌腱，去除大块肌肉。沿大腿根部朝上剪断（不要剪断大腿骨，漏出骨髓腔）。5. 用手剥离剩余的肌肉，直至露出干净的骨头。放在PBS中浸泡。6. 将骨头在75%的酒精中浸泡2分钟，用PBS清洗2次。再放置于PBS中。7. 将干净的剪刀，镊子，还有浸泡在PBS中的骨头用纸包好带到细胞房内。8. 剪断骨头两端，用10ml的注射器吸取1640，更换小针头。插入骨髓腔将细胞冲洗出，直至骨髓腔发白。9. 吹打细胞（近百下）直至细胞吹散。10. 用放置在50ml管上的40nm滤网过滤除去残渣，1300rpm/10min离心。11. 倒掉上清，加入BMDM培养基（加入量根据铺盘数确定），吹打细胞（近百下）直至细胞吹散。12. 吸取少量计数，若裂红，按照1106个/ml铺盘至15cm dish；若没裂红按照2106个/ml铺盘（冲洗出的细胞中有大量的红细胞）。13. 第4天：细胞开始大量贴壁（高吸附培养皿），更换BMDM培养基。此后每天加入2ml的新鲜BMDM培养基。长至第7天成熟。（若使用低吸附培养皿，最好裂红，因为macrophage不易贴壁，会有很多细胞悬浮，不裂红，红细胞会影响macrophage生长。而使用高吸附培养皿，在第4天换液时会把红细胞一起除去）14. 第7天，BMDM完全成熟，用胰酶消化1分钟，然后吹下细胞，计数后2-3106个/ml铺板做western。1-2106个/ml铺板收RNAIsolation of Mouse Bone Marrow Cells:1. Sacrifice mouse by cervical dislocation2. Clip the skin mid-back and remove the skin from the lower part of the body.3. Remove tissue from legs with scissors and dissect away from body.4. Clean remaining tissue from the femora and tibia and separate at knee joint. It is important to make sure that all the tissue is removed from the bones since cells associated with this can contaminate the marrow preparation and potentially overgrow the macrophages.5. Cut off each end of bone.6. Using a 27G needle and a 5 cc syringe filled with 3 ml PBS (or HANKS), expel the bone marrow into a 50ml screw top Falcon tube.7. Pass the cells through a cell strainer (40-100 um).8. Centrifuge, 1000 RPM 5 minutes. Resuspend cells in 10ml Bone Marrow Medium.9. Bring sample volume to 40ml with bone marrow medium. Count cells. Adjust the cell suspension to a concentration of 11 x 106 cells/ml. (normally from one mouse, one can get enough cells for six 15cm plates)10. Culture the cells at 37C under 5% CO2 (Change culture medium if needed). After 6-8 days, almost all adherent cells are BMM.