细胞造就操纵流程(国外英文资料).docVIP

细胞培养操作流程(国外英文资料) The cell transfer process Cell recovery: 1. Put fresh medium at 37 ℃ water bath pot temperature back, back to the temperature after spraying with 75% alcohol and wipe it, moved to the bench. Configuration: 50mL complete medium (10% FBS, 1% green streptomycin) 44.5 mL base medium + 5mL FBS + 0.5 mL green streptomycin double resistance. 2. Wear protective gloves, frozen tube taken out of the liquid nitrogen tank, check whether the lid is tight, in 37 ℃ water trough rapid thaw immediately (avoid ice recrystallization and cause cell death), rocking gently freeze pipe to make it melt in 2 minutes, with 75% alcohol to wipe save tube outside, to the bench. 3. To add thaw slowly 1 ml of the cell suspension cell culture, bottle to add 10 ml bottle complete medium (dilution ratio of 1:10), mixed evenly, in 37 ℃, 5% CO2 thermostatic cultivation in the box. Take 0.1 ml of thawing cell suspension for survival test. (Sf9 cells grown in 27 ℃, dont need CO2) Generally speaking, most cells do not need to remove cryoprotectants (e.g. DMSO) immediately. To remove immediately, then to add thawed cells suspension containing 5-10 ml of medium centrifugal tube, centrifugal 1300 RPM, 5 minutes, remove the supernatant, add fresh medium, mixing, into the CO2 incubator culture. If the refrigerant is not removed immediately, change the culture medium after the defrost culture. Subculture: 1. 37 ℃ constant temperature culture about 48 hours, after being cells full of 80-90%, from cultivation box 75 cm2 bottle, pour out the culture medium. Add the 5mLPBS buffer to wash the residual serum and remove the buffer. 2. (no cell side) along the wall slowly add 1 ml of the pancreatic enzyme solution digestive cells about 1 min, gently shaking, residual pancreatic enzyme, pour out the culture bottle in the constant temperature box for about 1 min, take out the culture bottle gently stroking the cell wall of one side. Note: the best digestion temperature is 37 ℃, add after fluid percussi