Cell culture FAQ
1 How should the freezing tube be thawed?
After taking out the cryotube, it should be quickly thawed in a 37 °C water tank, gently shake the cryotube to melt it in 1 minute, and note that the water surface should not exceed the edge of the cryotube cover, otherwise it will be prone to pollution. When the chilled tube is removed from the liquid nitrogen drum, it must be safe to prevent the freezing of the freezing tube.
When the cell cryotube is thawed, should DMSO be removed immediately?
Except for a few cells that are specifically sensitive to DMSO, most cell lines (including suspension cells) should be placed directly into a culture flask containing 10-15 ml of fresh medium after thawing, and replaced next day. Fresh medium can be used to remove DMSO, thus avoiding the problem that most of the cells cannot grow or attach after thawing.
3 Can I use a medium different from the original culture conditions?
No. Each cell strain has its own specific and adapted cell culture medium. If the culture medium with different culture conditions is used suddenly, the cells are mostly unable to adapt immediately, and the cells cannot survive.
4 Can I use serum types different from the original culture conditions?
No. Serum is an extremely important source of nutrients in cell culture, so the type and quality of serum can have a significant impact on cell growth. Serum from different species varies in the amount or content of some substances or molecules, and errors in serum use often cause 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 use of words, please do not use. CS (calfserum) refers to calf serum. HS (horseserum) refers to horse serum.
6 should use 5% or 10% CO2 when culturing cells? Or no effect at all?
Most of the mediums use HCO3-/CO32-/H+ as the buffer system for pH, 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 NaHCO3 in the medium is 1.5 g per liter, cells should be cultured with 5% CO2.
7 When do I need to change the medium?
Depending on the cell growth density, or in accordance with the replacement time on the basic data of the cell line, the medium can be changed on time.
Is there any antibiotic added to the medium? Except in special screening systems, no antibiotics should be added to the medium under normal culture conditions.
9 The concentration of trypsin-EDTA used in the substituting of adherent cells? What should I do?
The trypsin-EDTA concentration generally used is 0.05% trypsin-0.53 mM EDTA.4Na. * Immediately after opening the bottle, it should be dispensed in 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.
10 How should suspension cells be treated in a subculture?
Generally, it is only necessary to continuously add fresh medium to the original culture flask to dilute the cell concentration. If the culture solution is too much, the mouth of the culture flask can be raised slightly until it cannot be accommodated. When the bottle is dispensed, take out a part of the culture medium containing the cells to another new culture flask, and add the fresh medium to the appropriate concentration, and repeat the above steps.
11 How much speed should the centrifugal rate be to centrifuge the general animal cells?
To recover animal cells, the rate of centrifugation is typically 300xg (about 1,000 rpm), 5-10 minutes, and too high a speed will cause cell death.
What is the seeding density of 12 cells? Inoculation can be carried out according to the inoculation density or the proportion of the dilution plate on the basic data of the cell strain. Too few cells or too much dilution is an important cause of cell growth.
What is the composition of the 13 cell freezing medium?
When the animal cells are cryopreserved, the freezing medium used in the Zui is uniformly mixed with 5-10% DMSO (dimethylsulfoxide) and 90-95% of the original medium for cell growth. Note: Due to the large amount of heat released during DMSO dilution, DMSO should not be added directly to the cell fluid and must be prepared before use.
What is the 14 DMSO grade and sterile filtration method?
The DMSO grade used for cryopreservation must be Tissueculture grade DMSO (such as SigmaD2650), which is itself sterile. Immediately after opening the bottle, it should be dispensed in a small amount in a sterile test tube and stored at 4 °C to avoid repeated freezing. Thawing causes cleavage of DMSO to release harmful substances and reduces the chance of contamination. To filter DMSO, a DMSO-resistant Nylon filter is required.
15 method of cryopreservation of cells?
Cryopreservation method 1: The frozen tube is placed at 4 ° C for 30-60 minutes → (-20 ° C for 30 minutes *) → -80 ° C for 16 to 18 hours (or overnight) → liquid nitrogen tank vapor phase long-term storage.
Cryopreservation method 2: The cryotube is placed in a programmable cooling machine with a programmed procedure to drop 1-3 ° C to -80 ° C per minute, and then stored in a liquid nitrogen tank for long-term storage. * -20 °C can not exceed 1 hour to prevent the ice crystals from being too large, causing a large number of cell deaths. You can skip this step and put them directly into the -80 °C refrigerator, but the survival rate is slightly lower.
How many cell concentrations should there be in the cell cryotube when 16 cells are to be cryopreserved?
The number of cells in the cryotube is generally 1x106 cells/mlvial, and the fusion tumor cells are preferably 5x106 cells/mlvial.
17 How to avoid cell contamination? The types of cell contamination can be divided into bacteria, yeasts, molds, viruses, 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 formulation are good ways to reduce pollution.
18 What should I do if my cells are contaminated with microbes?
Discarded after direct sterilization.
Can 19 cells contaminated with mycoplasma be observed abnormally by the naked eye?
No. Most cell lines contaminated with mycoplasma, except for highly experienced experts,
Can't tell the difference by its appearance.
What effect does 20 original contamination have on cell culture?
Mycoplasma contamination can affect almost any data on growth parameters, metabolism, and research of all cells. Therefore, before performing the experiment, it must be confirmed that the cells are mycoplasma-free, and the data of the experimental results are meaningful.
21 What should I do if I detect cell line contamination with mycoplasma?
Discard after direct sterilization to avoid contamination of other cell lines.
How to keep the water tray of 22CO2 incubator clean?
Replace it regularly (at least every two weeks) with sterile distilled water or sterile deionized water.
23 Why is the medium stored in a 4 ° C refrigerator, the color will be dark red, and the pH will become more alkaline?
The medium is stored in a refrigerator at 4 ° C, and the CO 2 in the medium gradually overflows, causing the medium to become more alkaline. The color of the acid-base indicator (usually phenolred) in the medium will also be darker red as the alkalinity increases. As a result of the alkaline medium, the cell growth will be stagnant or dead. If the medium is alkaline, the sterile filtered CO2 can be passed to adjust the pH.
24 Are the fishes and flasks used for various cell cultures the same?
Different brands of dish or flask, the coating polymer is different, the manufacturing process is different, although it does not have much influence on most cells, only a few cells may have significant growth due to the use of different brands of dish or flask. difference.
25 After the cell cryotube purchased, after thawing, why is the number of cells too small?
The researchers showed that the number of cells in the culture of frozen cells was too small, mostly due to operational errors in the centrifugation process, resulting in physical damage to the cells and loss of cells. It is recommended that the cells should not be centrifuged immediately after thawing, and the medium should be replaced after the cells are grown overnight.
26 Possible reasons for cell death or poor cell survival?
Researchers have a poor survival rate in cell culture. Common causes can be summarized as: incorrect use of the medium or poor quality of the medium. The serum is used incorrectly or the serum is of poor quality. The thawing process is wrong. After the frozen cells are thawed, the cells are washed and centrifuged. Suspended cells are mistaken for dead cells. The culture temperature is incorrectly used. The cells were placed at –80 °C for too long.
27 The cork tube that was received was broken, the bottle cap was cracked, or the cap was peeled off?
Cracks in the chilled tube cap, or the rupture of the bottle, may be caused by improper force when the operator grips the cryotube, causing the cryotube to be cracked. It is recommended to use a hemostatic forceps to carefully grasp. In addition, the frozen cap is loose or loose, which is due to the physical phenomenon of thermal expansion and contraction. The cryotube may cause cell contamination. Therefore, when the cryotube is placed and removed, the cryotube should be immediately removed. Twist the Beijing equation creature once:
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