I. Operating procedures:

1. Before the experiment, irradiate the sterile room and sterile console with ultraviolet rays for 30-60 minutes for sterilization, wipe the sterile console with 70% alcohol, and turn on the fan of the sterile console for 10 minute, and then start the experiment operation. Treat only one cell at a time to avoid cross-contamination of cells. After the experiment, take the experimental items out of the table. If it is necessary to continue the next experiment, wipe the sterile console with 70% alcohol, and then let the fan of the sterile console run for 10 minutes before the next experiment can be carried out.

2. The working area for aseptic operation should be kept clean and spacious. Necessary items, such as test tubes, pipets or straws, can be temporarily placed, and other experimental items should be taken out in time after use to facilitate gas circulation. The experimental articles should be wiped with 70% alcohol before entering the aseptic operating table. The experimental operation should be carried out in the sterile area in the center of the console, not in the non-sterile area at the edge.

3. Carefully take out sterile laboratory supplies to avoid pollution. Do not touch the heads of straws and suction heads or the bottle mouth of the container, and do not operate the experiment directly above the open container. After the container is opened, hold the bottle cap by hand, hold the bottle body, tilt it about 45, and try not to put the lid of the bottle cap up on the table.

4. The staff should pay attention to their own safety, and must wear experimental clothes and gloves before the experiment. Special attention should be paid to human-derived or virus-infected cell lines, and an appropriate level of aseptic operating table (at least two levels) should be selected. During the operation, avoid generating aerosol, and be careful of toxic reagents, such as DMSO and TPA, to avoid sharp objects from injuring people.

5. Regularly check the following items: CO2 pressure in CO2 cylinder; CO2 concentration and temperature in the CO2 incubator, and whether the water tray is polluted; Whether the air pressure in the aseptic operation console is normal or not, replace the ultraviolet lamp tube, HEPA filter membrane and pre-filtration (300 hours/pre-filtration, 3000 hours /HEPA) regularly.

Second, the preparation of powder cell culture medium (taking 1L as an example):

Cell culture medium usually needs to add 10% serum, so the preparation volume of powder culture medium is 900 ml, and the pH is 7.2-7.4. Sodium bicarbonate was added separately. If sodium bicarbonate powder is directly added to the liquid culture medium, it will cause pH error or local overbased. Therefore, powder culture medium and NaHCO3 powder should be dissolved separately before mixing, and then CO2 gas should be used to adjust the pH, instead of strong acid (HCl) or strong alkali (NaOH), because chloride ion may affect the cell growth, and the pH value of the culture medium is easy to change during storage.

Reagents and equipment

1. purified water (milli-Q water or secondary or tertiary distilled water, the water quality is very important)

2. Powder media (1640, DMEM, etc. )

3. Sodium bicarbonate (Sigma S-40 19)

4. Electromagnetic stirrer

5. Sterile serum bottle

6.0.22 micron sterile filter membrane

7.pH meter

8. Vacuum valve

9. Carbon dioxide gas

Preparatory steps

1. dissolve the powder culture medium in 700 ml milli-Q water, and stir to dissolve it.

2. Weigh a proper amount of NaHCO3 powder (which varies according to the type of culture medium) and dissolve it in 200ml Milli-Q water, stir to dissolve it, and then introduce CO2 gas until it is saturated, about 3-5min.

3. Add the dissolved NaHCO3 solution containing saturated CO2 to the dissolved liquid culture medium and mix. The pH value of the mixed solution should be 7.2-7.4, unless the pH value deviation is too large, it is not necessary to adjust with acid and alkali. If the alkalinity is too strong, CO2 gas can be introduced to adjust the pH value .. When the culture medium passes through the filter membrane in vacuum, the pH value will increase by 0. 1-0.2.

4. Filter and sterilize with 0. 1 or 0.2 mm sterile filter membrane, and pack in sterile containers at the same time, indicate the type, date and bottle number of the culture medium, and store at 4℃ (serum can also be added to the culture medium for filtration).

5. The prepared culture medium must be tested for growth and pollution.

Matters needing attention

1. The liquid culture medium was stored in a refrigerator at 4℃ in the dark, and heated in a water tank at 37℃ before the experiment.

2. The storage period of liquid culture medium (including serum) is six months, during which glutamine may decompose. If the cells grow poorly, appropriate amount of glutamine can be added.