1. Before the experiment, the sterile room and the laminar flow are sterilized by UV lamp for 30-60 minutes, the aseptic operation is lifted with 70% ethanol, and the aseptic console fan is turned on for 10 minutes. After that, the experimental operation was started. Only one cell line was processed per operation, and the medium was not shared even if the medium was the same to avoid misunderstanding or intercellular contamination. After the experiment, the test article was taken out of the workbench and the aseptic operation was carried out with 70% ethanol. The operation interval should be such that the aseptic table is operated for more than 10 minutes before proceeding to the next cell line.

2. The aseptic operation work area should be kept clean and spacious. The necessary items, such as the test tube rack, straw suction device or straw box, can be temporarily placed. Other laboratory supplies should be removed after use to facilitate the circulation of airflow. The test article was wiped with 70% ethanol before being brought into the aseptic workstation. The experimental procedure should be in the central sterile area of ​​the lifting surface and not in the non-sterile area of ​​the edge.

3. Carefully use sterile laboratory items to avoid contamination. Do not touch the tip of the pipette or the mouth of the container, and do not operate the experiment directly above the open container. After the container is opened, hold the bottle cap with your hand and hold the bottle body, and use it at an angle of about 45°. Try not to place the cap on the table with the cap facing up.

4. Staff should pay attention to their own safety and must wear lab coats and gloves before conducting experiments. Special care should be taken for cell lines from human or viral infections and an appropriate level of aseptic table (at least Class II) should be selected. During the operation, avoid the generation of aerosol, beware of toxic drugs such as DMSO and TPA, and avoid damage from sharp needles.

5. Regularly check the following items: 5.1. CO2 pressure of CO2 cylinders 5.2. The CO2 concentration, temperature, and water tray of the CO2 incubator are contaminated (the water in the water tray is filled with sterile water and replaced weekly). 5.3. Airflow pressure in the aseptic operating station, regular replacement of UV lamps and HEPA filter membranes, pre-filter (300 hours / pre-filter, 3000 hours / HEPA).

6. Disinfectant (Zephrin 1:750) can be added to the sink, and the water in the sink should be replaced regularly.

Does the working concentration of trypsin should be stored at -20 degrees? If it is placed in a 4 degree refrigerator, how long can it be stored? Will it lose activity in a few hours?

Usually placed at -20 degrees, divided into 50 ml screw tube, when used, take a tube for 4 degrees.

1. Carry out the experiment carefully according to the operating procedures, which generally does not cause pollution. In many cases, the experiment fails due to contamination of the reagent or medium used.

2. After the powder medium is prepared (with serum), generally do not exceed 1 month at 4 degrees. For example, the storage time at -20 degrees may be longer, but it is better not to exceed 3-4 months. The requirements for immortalized cell lines are not too high, but I have been doing primary cell culture for the past 4 years, and the cells are delicate. Experience has shown that the placement time should not be too long.

3. Regarding the cleaning and disinfection of laboratory supplies, my experience is: used glassware should be soaked in clear water for more than 30min, then add a little cleaning agent and ultrasonically wash for about 30min (if there is no ultrasonic brush, soft brush can be used) Gently brush clean), remove and dry, then soak for 6-18h (or overnight) in the chromic acid wash solution, wash the tap water 10-15 times, double steamed water for 3-4 times, dry, after autoclaving Ready to use.

Many plastic products can also be autoclaved, such as culture caps, rubber stoppers, tips, and the like. Can not be used for high pressure has generally been made into a one-off product, of course, if the money is limited, such as imported culture plates, dishes, etc., can also be reused 1-2 times, I used to clean it completely It can be opened for 1-1.5 hours under UV light before use. I have done it many times without problems. Plastic culture bottles are inconvenient to clean and disinfect, it is best not to reuse, if it must be repeated, can be disinfected with ethylene oxide, etc. (it must be placed for more than half a year after disinfection)

Under light microscopy, epithelial cells are usually arranged in a "paving stone"-like manner, and there is a drawing phenomenon between the cells (that is, cells that are farther away can be connected by elongated tentacles), and the cells have the characteristics of growth. The interstitial cells are usually fusiform, with no growth characteristics, and the cells are not closely connected. However, the morphological characteristics of the cells may change due to changes in growth conditions. For example, HeLa cells are epithelial cells, but become fusiform under acidic culture conditions.

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