原代细胞培养和传代培养的方法及其注意事项(国外英文资料).doc

原代细胞培养和传代培养的方法及其注意事项(国外英文资料) Original culture The principle of ? ? The animal body tissue removed from the body, the enzymes (trypsin) is commonly used, chelating agent (EDTA) is commonly used or mechanical treatments, scattered into a single cell, suitable culture medium, the cell survival, growth and reproduction, a process called generation in primary culture. ? ????? Instruments, materials and reagents ? ? Instruments: incubator (adjust to 37 ℃), culture bottle, penicillin bottle, small glass funnel, AGAR, straws, pipette, gauze, surgical instruments, blood count plate, centrifuge, water bath (37 ℃) ? ? Material: animal tissue block ? ? Reagent: 1640 medium (containing 20% calf serum), 0.25% pancreatic enzyme, Hank s liquid, iodine The initial digestion method Prepare: take all kinds of disinfected culture products on the purifying mesa, and uv disinfection for 20 minutes. Wash your hands before you start, and rub your hands down to the elbow with 75% alcohol. Layout: light an alcohol lamp and install a straw hat. Treatment organization: place the block in a beaker and rinse it two or three times with the liquid. If you suspect that the tissue may be contaminated, you can put it in a mixture containing the green streptomycin for 30 to 60 minutes. Cut: cut the tissue from two to three millimeters in the eye of the eye for digestion. Add 30 to 50 times more trypsin fluid than the total amount of tissue, then pour into the triangle flask, and tie the jar or plug. 5. Digest: with the constant temperature water bath, or at 37 ℃ thermostat to digest, absorb every 20 minutes should shake once, such as electromagnetic thermostat blender better digestion. The digestion time depends on the size of the block and the stiffness of the organization. 6. The separation: in the process of digestion seen digestive juice opacity, sipping a bit digestive juices available in the microscopic observation, such as organization has dispersed into cells, or a single cell, immediately pu