1 How should the freezing tube be thawed? After removing the cryotube, it should be quickly thawed in a 37 °C water tank. Gently shake the cryotube to melt it in 1 minute. Note that the water surface should not exceed the edge of the cryotube cover, otherwise it will be contaminated. When the chilled tube is removed from the liquid nitrogen drum, it must be safe to prevent the freezing tube from bursting. 2 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 suspended 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 medium is different from the one originally provided, the cells are largely unable to adapt immediately, resulting in the cell not being able to 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 incorrect use of serum often causes cells to fail to survive. 5 What is FBS, FCS, CS, HS? FBS (fetal bovine serum) and FCS (fetal calf serum) have the same meaning, both refer to fetal bovine serum, FCS is the wrong use of words, please do not use. CS (calf serum) refers to calf serum. HS (horseserum) refers to horse serum. 6 Should 5% or 10% CO2 be used when culturing cells? Or not at all? Most of the mediums use HCO3-/CO32-/H+ as the pH buffer system, and the NaHCO3 content in the medium will determine the CO2 concentration 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 according to the replacement time on the basic data of the cell line, the medium can be changed on time. 8 Do you need to add antibiotics to the medium? Except in special screening systems, no antibiotics should be added to the medium under normal culture conditions. 9 What is the trypsin-EDTA concentration used for the attachment of adherent cells? What should I do? The trypsin-EDTA concentration is generally 0.05% trypsin-0.53mMEDTA.4 Na. Immediately after the first bottle opening, it should be dispensed in a small amount in a sterile test tube and stored at –20 °C to avoid repeated freezing and thawing to reduce the activity of trypsin and reduce the chance of contamination. 10 How should suspended 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 is unable to be accommodated. When the bottle is dispensed, take out a part of the culture medium containing the cells to another new culture flask, add the fresh medium to the appropriate concentration, and repeat the above steps. 11 What is the speed at which the centrifugal rate should be centrifuged? To recover animal cells, the centrifugation rate is typically 300xg (about 1,000 rpm), 5-10 minutes, and the high speed will cause cell death. 12 What is the density of cells inoculated? 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. 13 What is the composition of the cell freezing medium? The most commonly used freezing medium for cryopreservation of animal cells is a homogeneous medium containing 5 - 10% DMSO (dimethyl sulfoxide) and 90 - 95% of the original cells 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. 14 What is the level of DMSO and the way to sterile filtration? The DMSO grade used for cryopreservation must be Tissue culture grade DMSO (eg Sigma D2650), which is itself sterile. Immediately after the first opening, it should be dispensed in small quantities in sterile tubes and stored at 4 °C. Avoid repeated freezing and thawing to cause DMSO cracking to release harmful substances and reduce the chance of pollution. To filter DMSO, a DMSO-resistant Nylon filter is required. 15 How to cryopreserve cells? Cryopreservation method 1: The cryotube is placed at 4 °C for 30~60 minutes → (-20 °C for 30 minutes*) → -80 °C for 16~18 hours (or overnight) → Liquid nitrogen tank vapor phase for 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 should 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 some. 16 How many cell concentrations should cells have in the cryotube when the cells are to be cryopreserved? The number of cells in the cryotube is generally 1x106 cells/ml vial, and the fusion tumor cells are preferably 5x106 cells/ml vial. 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 media formulation are the best ways to reduce contamination. 18 What should I do if my cells are contaminated with microbes? Discarded after direct sterilization. 19 Mycoplasma Contaminated cells can 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 mycoplasma pollution 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 my cell strain is detected to be contaminated with mycoplasma? Discard after direct sterilization to avoid contamination of other cell lines. 22 How can the water tray of the CO2 incubator be kept 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 CO2 in the medium gradually overflows, causing the medium to become more alkaline. The color of the acid-base indicator (usually phenol red) 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 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 have much influence on most cells, only a few cells may have significant growth due to the use of different brands or flasks. difference. 25 After the thawed cell cryotube purchased, 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 once. Another: Here to add disinfection of culture supplies, for reference: The greatest risk of cell culture is the contamination of bacteria, fungi and viruses in the culture. The pollution is mainly caused by the operator's negligence. The common causes are the uncleanness of the operation room or the surrounding space. The culture vessels and culture fluids are not disinfected. Qualified or incomplete, because the failure of each link in the culture can lead to failure of culture, every step of cell culture should strictly abide by the operating rules to prevent pollution. There are three types of disinfection methods: (A) Physical sterilization (ultraviolet rays, moist heat, slag, etc.); (B) Chemical sterilization (various chemical disinfectants); (C) Antibiotics. (1) UV disinfection: used for air, surface of the console and cannot be disinfected and cultured using other methods. Direct ultraviolet radiation is convenient and effective. After a certain period of time, most of the bacteria in the air can be eliminated. The ultraviolet light in the culture room should not exceed 2.5 meters from the ground, and the disinfected items should not be shielded from each other. Less than disinfection. Ultraviolet rays can produce ozone, pollute the air, reagents and culture fluids have adverse effects, and also cause damage to human skin. It is not suitable for near-irradiation experiments. (2) Warm disinfection: high pressure steam disinfection is the most widely used and best disinfection method. When warming and disinfecting, the disinfectant items should not be overfilled to prevent gas clogging in the sterilizer and millions of dangers, ensuring the circulation of gas inside. Before heating and boosting, first open the exhaust air to discharge the cold air in the sterilizer. After the cold air is exhausted, close the exhaust valve and check that the safety valve is free to move, then start to increase the pressure. When the required pressure is reached, Start to calculate the disinfection time. During the disinfection process, the operator cannot leave the job, and should regularly check the pressure and safety to prevent disinfection and epidermal accidents. Disinfection pressure and time of common items: Culture medium, rubber products, 10 pounds for 10 minutes; Cloth, glassware, metal equipment, 18 pounds and 20 minutes. The top two are the most common physical disinfection methods. (3) Chemical disinfection method: The most common is 70% alcohol and 1 ‰ clean and dry, the former is mainly used for the operator's skin, the surface of the console and the wall of the sterile room. The latter is mainly disinfected by soaking of the instrument and wiping the skin of the skin and the operating room. The chemical disinfection method is simple, convenient and effective. (4) Antibiotic disinfection: It should be recorded as antibiotic sterilization, mainly used for sterilizing culture liquid or preventing culture pollution. If fungal contamination has occurred, it should be discarded as soon as possible. However, if it is a precious cell line, it can be tried with antifungal drugs. It can be treated with 5-10 times of the general dosage. After 24 to 28 hours, the conventional culture solution is changed. . But this method is generally only effective in the early stages of pollution! 35. Does divalent ion inhibit trypsin activity? What is the purpose of adding EDTA when using trypsin? Divalent ions do inhibit trypsin activity. EDTA is used to chelate free magnesium and calcium ions in order to maintain trypsin inhibition. It is recommended to wash the cells with EDTA before trypsinizing the cells to eliminate all divalent ions from the medium. 44. Can serum and antibiotics be added to the medium for long-term storage? Once you have added serum and antibiotics to your fresh medium, you should use it within two to three weeks. Because some antibiotics and essential components in serum begin to degrade after thawing. 45. Can the medium and other additives and reagents be freeze-thawed repeatedly? Most additives and reagents can freeze and thaw up to 3 times. If the number of times is more, it will cause a certain level of degradation and precipitation in the protein-containing solution, which will affect its performance. 46. ​​Why use a liquid trypsin solution stored in a 4 ° C refrigerator for one week of dissolution? Trypsin may begin to degrade at 4 ° C and become unstable if left at room temperature for more than 30 minutes. 47. The best way to preserve serum? We recommend that serum should be stored at -5 ° C to -2 ° ° C. Do not exceed one month when stored at 4 °C. If it is not possible to use one bottle at a time, it is recommended that the serum be aseptically packed into a suitable sterile container and returned to the freezer. 48. How to thaw serum will not damage the quality of the product? After the serum was taken out of the freezer, it was first placed in a refrigerator at 2 to 8 ° C to be melted, and then completely melted at room temperature. However, it must be noted that the melting process must be regularly shaken evenly. 49. After the serum is thawed, flocculent deposits are found. What should I do? There are many reasons for the presence of precipitates in serum, but the most common cause is due to the degeneration of lipoproteins in the serum, and fibrin (one of the proteins that form blood coagulation) is also present in the serum after serum thawing. It is also one of the causes of sedimentation. However, these flocculent precipitates do not affect the quality of the serum itself. To remove these flocculent deposits, the serum can be dispensed into a sterile centrifuge tube and centrifuged slightly at 400 g. The supernatant can then be added to the medium for filtration. It is best not to use a filtration method to remove these flocculent deposits as it may clog the filter membrane. 50. Why do you want to heat inactivate serum? Heating can inactivate the complement system. Activated complement is involved in lytic cell events, stimulates smooth muscle contraction, releases histamine from cells and platelets, and activates lymphocytes and macrophages. In immunological studies, it is recommended to use heat-inactivated serum when culturing ES cells and smooth muscle cells. 51. Is it necessary to do heat inactivation? Experiments have shown that properly treated heat inactivated serum is not required for most cells. The serum thus treated has only a slight promotion to the growth of the cells, or has no effect at all, and even the high temperature treatment usually affects the quality of the serum, resulting in a decrease in the cell growth rate. The heat-treated serum, the formation of precipitates will increase significantly, these precipitates observed under an inverted microscope, such as "small black spots", often let researchers mistakenly believe that serum is contaminated, and put serum in 37 In the °C environment, this precipitate will be increased, which makes the researchers mistakenly believe that it is the splitting and amplification of microorganisms. If not necessary, you do not need to do the heat treatment step. Not only save time, but also ensure the quality of serum! 52. Why does fetal bovine serum stored in the refrigerator precipitate? Some fetal bovine serum products are not pre-aged. When stored at 2-8 ° C, various proteins and lipoproteins in serum (such as cold agglutinin, fibrinogen, villogen, etc.) may aggregate to form precipitates or visible turbidity. . This should not affect the quality of the serum. It is recommended to store fetal bovine serum at -20 °C to avoid repeated freezing and thawing. 53. How to avoid the formation of sediment? We recommend that you take note of the following when using serum: (1) When thawing serum, please follow the recommended step-by-step thawing method (-20 ° C to 4 ° C to room temperature). If the temperature is too large when the serum is thawed (eg -20 ° C to 37 ° C), it is very easy to produce sediment. . (2) When thawing serum, please shake it at any time to make the temperature and composition uniform, and reduce the occurrence of sedimentation. (3) Do not leave the serum at 37 °C for too long. If left at 37 ° C for too long, the serum will become cloudy, and many of the more unstable components in the serum will be damaged, which will affect the quality of the serum. (4) The heat inactivation of serum is very likely to cause an increase in sediment, and this step is not necessary if it is not necessary. (5) If heat inactivation of serum is necessary, please observe the principle of 56 ° C, 30 minutes, and shake it at any time. If the temperature is too high, the time is too long or the shaking is uneven, it will cause an increase in sediment. Sunflower Seeds 363,Sunflower Seeds Type 363,Giant Sunflower Seeds,Edible Sunflower Seeds Inner Mongolia Hengxintonghui Supply Chain Management Services Co.,LTD , https://www.hxthfood.com