1. Received cells, please check if the bottle has ruptured, if the culture medium has leaked, and if it is turbid. If so, please contact us as soon as possible.
2. Received cells, if the packaging is intact, please observe the cells under a microscope. Due to transportation issues, adherent cells in the cell culture bottle may detach from the bottle wall, causing cell suspension under a microscope. When this occurs, please do not open the cell culture bottle and do not aspirate the culture medium first. The culture bottle should be immediately placed in a cell culture incubator and left to stand for about 3-5 hours, allowing the cells to stabilize before observing under a microscope. At this point, most cells will reattach to the bottle wall. If the cells still cannot adhere to the wall, please use trypan blue staining to identify cell viability. If trypan blue staining confirms normal cell viability, please handle it according to the suspension cell method
3. If there are no abnormal conditions when receiving the cells, please observe the cell density under a microscope. If it is a adherent cell and the confluence degree does not exceed 80%, use 75% alcohol (it is recommended to prepare water with 75% alcohol, which is also sterilized ultrapure water). Spray the entire bottle for disinfection and place it in the ultrabacterial table. Strictly follow the aseptic operation: then open the lid, first use a gun to suck out about 10ML of the culture medium, put it in a centrifuge tube, and then burn the bottle mouth (it is strictly prohibited to pour it directly), which can ensure no contamination. Then use a 10ML pipette to suck out the remaining culture medium and place it in the centrifuge tube. Only about 5-10ML of culture medium is left in the culture bottle for further cultivation. When the confluence degree exceeds 80%, please follow the cell culture conditions. Passage cultivation.
If it is a suspended cell, aspirate the culture medium, centrifuge at 1000 rpm for 3-5 minutes, aspirate the supernatant, suspend the cells in fresh culture medium at the bottom of the tube, and then transfer them back to the culture bottle.
4. Place the culture bottle in a 37 ℃ incubator and gently loosen the lid. The extracted culture medium can be stored in sterilized bottles and kept in a 4 ℃ freezer for future use. When changing the medium the next day, fresh culture medium and imported fetal bovine serum should be prepared. This is better for cell growth. Don't use our original bottle of culture medium anymore.
5. After 24 hours, the cell morphology has recovered and covered the bottle wall, and it can be passaged. (adherent cells) Pour out the culture medium from the culture bottle, add 3-5ml (based on the coverage of the cell growth surface) of PBS or Hanks' solution, wash and discard. Add 0.5-1ml of 0.25% EDTA containing trypsin for digestion, with digestion time depending on the specific cell, generally 1-3 minutes, not exceeding 5 minutes. It can be digested in a 37 ℃ incubator. Gently shake the bottle wall until the cells fall off, then add 3-5ml of culture medium to terminate digestion. Gently blow the cells on the bottle wall with a pipette to completely detach them, then draw the solution into a centrifuge tube and centrifuge at 1000rpm for 5 minutes. Discard the supernatant and determine the number of bottles based on the number of cells. Generally, one bottle is passed to two, and if there are a large number of cells, three bottles can be passed to one. Some cells are not easily transmitted too thinly, while others that grow faster can be passed to several bottles, depending on the specific cells and experience. (Suspended cells) Gently blow the bottle wall with a pipette and directly draw the solution into a centrifuge tube for centrifugation.
6. Adhering cells and suspended cells. Strict aseptic operation. When changing the medium, replace with a new cell culture bottle, fresh culture medium, and imported fetal bovine serum, and culture at 37 ° C with 5% CO2
After receiving the cells, please observe them under a microscope and handle them in an appropriate manner. If there are many suspended cells, please collect them by centrifugation and inoculate them into a new culture bottle. Discard the original solution, use freshly prepared culture medium, and use imported fetal bovine serum Just received the cells, if there are not many cells, the serum concentration can be increased to 15% for cultivation. If the cell count reaches around 80%, the serum concentration remains at 10%.
Cell culture conditions
11640 medium+10% imported fetal bovine serum+2 mM L-G (L-glutamine)+1 mM Sodium pyrovate+10 mM HEPES (gibbco.). Item number. 15630-080)
2. DMEM high glucose medium+10% imported fetal bovine serum+4 mM L-G (L-glutamine)+1 mM (sodium pyruvate 1, HEPES
3. E, MEM medium+10% imported fetal bovine serum+NEAA (non essential amino acids)+2 mM L-G (L-glutamine)+1 mM (sodium pyruvate)
4. F12K medium+10% imported fetal bovine serum+2 mM L-G (L-glutamine)
5. McCoy's 5a medium+10% imported fetal bovine serum+1.5mM L-G (L-glutamine)
6 . L 15 medium+10% imported fetal bovine serum (GIBCO, item number 10099141)+1.5 mM L-G (L-glutamine)
7. DMEM/F12 medium+10% imported fetal bovine serum+1.5 mM L-G (L-glutamine)
8. IMDM medium+10% imported fetal bovine serum+1.5 mM L-G (L-glutamine)
2. L-G (L-glutamine). L-Glutamine 200mM
3. NEAA (Non essential Amino Acid)
4..sodium.pyruvate
100ML 1640 complete culture medium preparation method
1640 medium 86ML
FBS serum 10ML
L-Glutamic acid amine. 100X 1ML
Sodium pyruvate 100X 1ML
HEPES(1M) 10mM 1ML
Antibiotics (penicillin, streptomycin) 1ML
