Cell culture medium method

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= cell culture medium method =
 
(1) Preparation and installation of filters (2) Preparation of synthetic medium (3) Treatment of calf serum (4) Preparation of growth medium
(5) Recovery of cryopreserved cells (6) Passage (7) Frozen storage (8) Precautions

(1) Prepare and install filters
The filter was cleaned, dried, and placed in a microporous membrane with a pore size of 0.22 μm. It was packed in a cloth and autoclaved at 15 lbs. 20 min. Open the filter holder in the clean bench. One end of the hose is connected to the filter pump and inserted into the liquid to be sterilized. The outlet end hose is deep into the sterilized bottle. Filter with a pump and check if the filter is intact after filtration.

(2) Preparation of synthetic medium
1. According to the instructions in the cell catalogue, select the appropriate dry powder of the culture medium according to the following table and use the appropriate amount of ultrapure water to dissolve.
Medium Brand Item number
D-MEM/F-12 GIBCO 12400024
Dulbecco's Modified Eagle Medium (D-MEM), powder (high glucose) GIBCO 12800017
F-12 Nutrient Mixture (Ham) powder GIBCO 21700075
Iscove's Modified Dulbecco's Medium (IMDM) powder GIBCO 12200036
Leibovitz's L-15 Medium powder GIBCO 41300039
McCOY's 5A SIGMA M4892
Minimum Essential Medium (MEM) Alpha Medium powder (MEMα)
With ribonucleosides and deoxyribonucleosides 		GIBCO 11900024
Minimum Essential Medium (MEM) Alpha Medium powder (MEMα)
Without ribonucleosides and deoxyribonucleosides 		GIBCO 12000022
Minimum Essential Medium (MEM) powder GIBCO 41500034
NUTRIENT MIXTURE F12 HAM KAIGHN'S
MODIFICATION (F12K) 		SIGMA N3520
RPMI Medium 1640 GIBCO 31800022
EGF SIGMA E9644
Sodium pyruvate SIGMA P2256-25G
MEDIUM 199, WITH EARLE'S SALTS AND L-GLUTAMINE, WITHOUT SODIUM BICARBONATE. CELL CULTURE TESTED SIGMA M5017

2. Add sodium bicarbonate, sodium glutamate, HEPES, etc. as required, and stir well to dissolve.
3. Adjust the pH to about 7.2.
4. Add water to the final volume.
5. Filter and sterilize the solution in a clean bench, place it in a 250 mL or 500 mL glass vial, and tighten the bottle with a flip cap.
6, the bottle mouth is sealed, stored in a refrigerator at 4 °C.

(3) Treatment of calf serum
The calf serum sold on the market is generally sterilized, but it should be heat inactivated before use, that is, the method of heating destroys the complement (there is also a view that heat inactivation treatment is unnecessary). Fetal bovine serum does not have to be inactivated.
1. Heat the serum to 56 ° C for 30 min, and gently shake it from time to time to make the heat even and prevent precipitation.
2. The treated serum was stored at 4 °C.
3, calf serum is best screened before use to master the quality of serum.

(4) Preparation of growth medium
In addition to serum-free culture, various synthetic media require the addition of a certain amount of calf serum or fetal bovine serum and antibiotics prior to use.
  1. The medium was dispensed into vials (100-200 mL) for use, and the cap was stoppered to close the mouth.
  2. It is prepared according to the following proportions: basic medium accounts for 80%~90%, and calf serum or fetal bovine serum accounts for 10%~20%. The double-antibody stock solution (penicillin + streptomycin) was added in a volume fraction of 1% so that the final concentrations of penicillin and streptomycin were 100 U/mL and 100 μ/mL, respectively.
(5) Recovery of cryopreserved cells
  1. The principle of slow freezing and fast melting should be observed. First adjust the water bath to 37-37. 5 degrees, take out the frozen cells and quickly put them into the surface of the water and shake them until they melt.
  2. Add the complete medium to a 50ml small flask in a sterile table, about 5ml, then remove the cells from the cryotube with a sterile pipette, shake it gently, and shake the cells evenly. .
(6) Passage:
  1. Adherent cells:

    For adherent cells, the medium should be sucked (pour) first, and the cleaner the better, so as not to add the digestive juice after neutralization, so that the strength is weakened (or 1-3 washes with PBS). 50ml culture flask is added to the digestive juice about 1-3ml, digested according to this ratio, (according to experience), shaking the digestive solution evenly placed in a 37-degree incubator for about 2-5 minutes, under the microscope, see the cell shrinking round or a few falling off Gently vibrate the bottom of the bottle to completely detach the cells. After adding 2-3ml of complete medium, gently pipette, so that the cells are basically suspended in a single suspension, then separate into other sterile culture flasks, and continue to culture or experiment after adding complete medium. .

  2. Suspension cells:

    Generally, the cell stock solution can be directly placed in other culture flasks, and the culture medium can be added to continue the culture. If the concentration is high, the centrifuge can be centrifuged at 1000 rpm. After 5 minutes, the complete medium is added. After gently blowing, the other culture flasks are added. Complete medium is continued to culture.

(7) Freezing

The adherent cells were digested, collected by centrifugation, and the suspended cells were directly collected by centrifugation, and the cells were resuspended in complete medium or fetal bovine serum to a final concentration of about 106/ml. Add 10% DMSO. Pack 1 to 2 ml per tube into the cryotube. Wrap in a refrigerator at -70 ° C overnight with a heat-insulating material. Save to liquid nitrogen the next day.

(eight) matters needing attention
  1. Disinfection of glass pipettes and glass culture bottles: 1) autoclave sterilization for more than 15 minutes; 2) dry sterilization and disinfection for 140 degrees for more than 2 hours;
  2. The sterile workbench is first cleaned and then wiped clean with 75% alcohol, UV irradiation for more than 40 minutes; various culture plates are irradiated for more than 3 hours;
  3. After the medium (pH 7.2) and the serum are prepared, the sterility test should be carried out: the serum is added to the medium at 10%, and the complete medium is cultured in a sterile glass centrifuge tube or a glass bottle to take 5-10 ml. For 2-3 days, the naked eye sees no foreign matter such as turbidity or sedimentation. Store at 4 degrees after dispensing;
  4. Digestion solution (pH 7.8) or other addition solution, using high pressure steam sterilization or disposable sterile filter to filter and sterilize, and pack it into a -20 degree storage;
  5. The incubator should be cleaned with water and then wiped with 75% alcohol (if there is ultraviolet light, it should be irradiated for more than 1 hour, if there is high temperature sterilization, it should be programmed according to the procedure). At least once a month.
  6. When entering the operation, be careful to wash your hands and wrists first, then wipe with 75% alcohol. Pay attention to the spatial level of the aseptic table during operation. Hand and items should not be placed above the exposed bottle mouth. If the quantity is large, the culture bottle should be placed in parallel with the alcohol lamp for easy operation. The bottle and the bottle should be separated by a certain distance. When opening the bottle (cover), use it first. 75% alcohol is repeatedly wiped or burned with a lamp. After opening, apply the lamp to burn the mouth first, then burn the lid. Do the same after using it. The entire operation process should be as far as possible inside the sterile table.

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