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After removing the freezing tube, immediately place it in a 37 ° C water tank for quick thawing. Gently shake the freezing tube to melt it within 1 minute, and pay attention that the water surface should not exceed the edge of the freezing tube, otherwise pollution may occur. In addition, when the freezing tube is taken out of the liquid nitrogen barrel and thawed, it is necessary to pay attention to safety and prevent the explosion of the freezing tube.
2 Should the DMSO be removed immediately when the cell cryotube is thawed?
Except for a few cells that are specifically indicated to be sensitive to DMSO, most cell lines (including suspended cells) should be placed directly into culture angle flasks containing 10-15 ml of fresh medium after thawing, and replaced after the next day Fresh medium can be used to remove DMSO, so as to avoid the problem that most cells cannot grow or adhere after thawing.
3 Is it possible to use media with different conditions from the original culture?
Can't. Each cell line has its own specific and adapted cell culture medium. If the culture medium used is different from the original culture conditions, most cells will not be able to adapt immediately, resulting in the cell's inability to survive.
4 Can I use different serum types from the original culture conditions?
Can't. Serum is an extremely important source of nutrients in cell culture, so the type and quality of serum will have a great impact on cell growth. Serum from different species differs in the amount or content of some substances or molecules. Incorrect use of serum often causes cells to fail to survive.
5 What is FBS, FCS, CS, HS?
FBS (fetalbovineserum) and FCS (fetalcalfserum) have the same meaning. Both refer to fetal bovine serum. FCS is the wrong word. Please do not use it again. CS (calfserum) refers to calf serum. HS (horseserum) refers to horse serum.
6 Should I use 5% or 10% CO2 when culturing cells? Or no effect at all?
Generally, HCO3- / CO32- / H + is used as the pH buffer system in the medium, and the content of NaHCO3 in the medium will determine the concentration of CO2 that should be used in cell culture. When the NaHCO3 content in the medium is 3.7 g per liter, 10% CO2 should be used for cell culture; when the NaHCO3 in the medium is 1.5 g per liter, 5% CO2 should be used to culture the cells.
7 When do I need to change the medium?
Depending on the cell growth density, or follow the replacement time on the basic data of the cell line, just change the medium on time.
8 Do I need to add antibiotics to the medium? Except in a special screening system, generally no antibiotics should be added to the medium under normal culture conditions.
9 The concentration of trypsin-EDTA used in the passage of adherent cells? What should I do?
The concentration of trypsin-EDTA generally used is 0.05% trypsin-0.53mMEDTA.4Na. After opening the bottle for the first time, it should be divided into a small amount in a sterile test tube and stored at –20 ° C. Avoid repeated freezing and thawing to reduce the activity of trypsin and reduce the chance of contamination.
10How should the suspension cells be subcultured?
Generally, it is only necessary to continue to add fresh medium to the original culture angle flask to dilute the cell concentration. If there is too much culture medium, the mouth of the culture angle flask can be raised slightly until it cannot be accommodated. When splitting the flask, take out a part of the culture solution containing cells to another new culture corner flask, add fresh medium to dilute to the appropriate concentration, and repeat the previous steps.
11 If you want to centrifuge general animal cells, what should be the centrifugal speed?
To recover animal cells, the centrifugal speed is generally 300xg (about 1,000 rpm), 5-10 minutes, excessively high speed will cause cell death.
What is the seeding density of 12 cells? Inoculate according to the inoculation density on the basic data of the cell line or the ratio of the dilution plate. Too few cells or too much dilution is also one of the important reasons why cells cannot grow.
13 What is the composition of the cell freezing medium?
The most commonly used freezing medium for animal cell cryopreservation is to mix 5-10% DMSO (dimethylsulfoxide) and 90-95% of the original medium used for cell growth. Note: Because DMSO will release a lot of heat energy when it is diluted, it is not possible to add DMSO directly to the cell fluid. It must be prepared before use.
14 What is the grade of DMSO and the method of sterile filtration?
The DMSO grade used for cryopreservation must be Tissueculturegrade's DMSO (such as SigmaD2650), which is itself aseptic. It should be packed in sterile test tubes immediately after the first opening, and stored at 4 ° C to avoid repeated Freezing and thawing causes DMSO to crack and release harmful substances, and can reduce the chance of pollution. To filter DMSO, it is necessary to use DMSO resistant Nylon filter membrane.
15 How to cryopreserve cells?
Cryopreservation method 1: Place the cryotube at 4 ° C for 30 to 60 minutes → (-20 ° C for 30 minutes *) → -80 ° C for 16 to 18 hours (or overnight) → liquid nitrogen tank vaporphase for long-term storage.
Cryopreservation method 2: Place the cryotube in the programmable temperature-reducing machine that has been set to a temperature below 1-3 ° C to –80 ° C per minute, and then put it into the liquid nitrogen tank vaporphase for long-term storage. * Do not exceed 1 hour at -20 ° C to prevent the ice crystals from becoming too large and causing a large number of cell deaths. You can also skip this step and put it directly in the -80 ° C refrigerator, but the survival rate is slightly reduced.
16 When cells are to be cryopreserved, what cell concentration should be in the cell cryotube?
The number of cells in the cryotube is generally 1x106cells / mlvial, and 5x106cells / mlvial for fusion tumor cells.
17 How to avoid cell contamination? The types of cell contamination can be divided into bacteria, yeast, mold, virus and mycoplasma. The main causes of contamination are improper aseptic technique, poor operating room environment, contaminated serum and contaminated cells. Strict aseptic technique, clean environment, and good quality cell source and medium preparation are the best ways to reduce pollution.
18 What should I do if the cells are contaminated with microorganisms?
Discard after direct sterilization.
Can the cells contaminated by mycoplasma 19 be visually observed?
Can't. Except for highly experienced experts, most cell lines that are contaminated with Mycoplasma,
It cannot be distinguished by its appearance.
How does 20 Mycoplasma contamination affect cell culture?
Mycoplasma contamination can affect almost all cell growth parameters, metabolism, and any data from research. Therefore, before conducting the experiment, you must confirm that the cells are mycoplasma-free, and the data of the experimental results are meaningful.
21 What should I do if my cell line is detected to be contaminated with mycoplasma?
Discard after direct sterilization to avoid contamination of other cell lines.
How to keep the water tray of 22CO2 incubator clean?
Change it regularly (at least every two weeks) with sterile distilled water or sterile deionized water.
23 Why is the medium kept in a refrigerator at 4 ° C, the color will be darker red, and the pH value will become more and more alkaline?
The medium is kept in a refrigerator at 4 ° C. The CO2 in the medium will gradually overflow, causing the medium to become more alkaline. The color of the acid-base indicator (usually phenolred) in the culture medium will become darker red as the alkalinity increases. If the medium is alkaline, it will cause cell growth to stagnate or die. If the medium is alkalescent, sterile filtered CO2 can be passed to adjust the pH.
24 Are the dishes and flasks used for various cell cultures the same?
Different brands of dish or flask have different coating polymers and different manufacturing procedures. Although they do not affect most cells, only a few cells may have significant growth due to the use of different brands of dish or flask difference.
25 After thawing the cell cryotube purchased, why does the number of cells become too small?
The researchers found that the number of cells was too small during the cultivation of frozen cells, mostly because of errors in the operation of the centrifugation process, resulting in physical damage to the cells and cell loss. It is recommended not to centrifuge the cells immediately after thawing, but to change the medium after the cells grow overnight.
26 Possible reasons for cell death or poor cell survival rate purchased?
Researchers experienced poor survival rates during cell culture. Common reasons can be summarized as: incorrect media usage or poor media quality. Incorrect use of serum or poor quality of serum. The thawing process is wrong. After the frozen cells are thawed, the cells are washed and centrifuged. Suspended cells are mistaken for dead cells. Incorrect culture temperature. Leave the cells at –80 ° C for too long.
27 The bottle body of the received cryotube is broken, the cap is cracked, or the cause of the cap falling off?
A crack in the cap of the cryotube, or a cracked bottle body, may be caused by the operator's improper force when gripping the cryotube, causing the cryotube to break. It is recommended to use a hemostatic forceps to carefully grip it. In addition, the cap of the freezing tube is loose or loose, due to the physical phenomenon of thermal expansion and contraction. The freezing tube may cause cell contamination. Therefore, when placing and removing the liquid nitrogen barrel, the freezing tube should be replaced immediately. Twist once.