Fishbone diagram is a quality management technique that analyzes the main causes of abnormal cell morphology, including microbial contamination, cell level, human operation/environment, culture medium, auxiliary reagents, consumables, etc. XP Biomed's technical support department also provides a series of solutions to help researchers solve experimental problems. According to the near to far of the fish bone and the size of the fish bone, the corresponding probability of the problem is from large to small, and the user can check the cause of the cell culture abnormality and countermeasures according to the map.
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Consumables -
Culture flasks1. The culture bottle belongs to the category of disposable replacement consumables, and repeated use may cause cell contamination or unstable results such as cell failure to adhere, aging, excessive activation, etc; 2. Plastic consumables may carry silicone oil when removed from the mold, which affects cell growth; 3. Most production consumables workshops are not clean workshops, and consumables may carry foreign objects.
1. The culture bottle belongs to the category of disposable replacement consumables, and repeated use may cause cell contamination or unstable results such as cell failure to adhere, aging, excessive activation, etc;
2. Plastic consumables may carry silicone oil when removed from the mold, which affects cell growth;
3. Most production consumables workshops are not clean workshops, and consumables may carry foreign objects.
Please investigate and select consumables with stable and qualified quality, and request the manufacturer to provide test data reports.
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Petri dishes/well platesThe bottom surface of culture consumables with poor quality may be uneven under microscopic magnification, resulting in inconsistent dispersion of cell adhesion and aggregation at the concave area.The bottom surface of culture consumables with poor quality may be uneven under microscopic magnification, resulting in inconsistent dispersion of cell adhesion and aggregation at the concave area.Please investigate and select consumables with stable and qualified quality, and request the manufacturer to provide test data reports.
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PipetteWhen dividing different cell lines, the gun head and pipette need to be replaced at once to avoid cross contamination.When dividing different cell lines, the gun head and pipette need to be replaced at once to avoid cross contamination.
As a low probability event, the probability of different types of consumables not reaching sterilization (i.e. possibly containing bacteria) ranges from 1/1000 to 1/1000000.
This type of low probability event that occurs randomly is the most difficult to track, investigate, and prevent.
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Auxiliary reagent -
AntibioticCells grow in a sterile state in the body, and conventional culture of cell lines should avoid adding antibiotics when not necessary. Antibiotics are known to have different effects on cells, such as: 1.one β- Lactam antibiotics (penicillin) - can affect protein synthesis and inhibit cell proliferation; 2.Aminoglycosides - Aminoglycosides (streptomycin, kanamycin, gentamicin) - can reduce protein synthesis, lower ATP levels, and produce cytotoxicity; 3.Lactones (amphotericin B/fungizone, nystatin) - interfere with cell membranes and produce cytotoxicity.Cells grow in a sterile state in the body, and conventional culture of cell lines should avoid adding antibiotics when not necessary. Antibiotics are known to have different effects on cells, such as:
1.one β- Lactam antibiotics (penicillin) - can affect protein synthesis and inhibit cell proliferation;
2.Aminoglycosides - Aminoglycosides (streptomycin, kanamycin, gentamicin) - can reduce protein synthesis, lower ATP levels, and produce cytotoxicity;
3.Lactones (amphotericin B/fungizone, nystatin) - interfere with cell membranes and produce cytotoxicity.When to use antibiotics:
1. During primary cell culture. Because it is easy to introduce pollution when separating from the organization;
2. When microbial contamination is observed and confirmed (after removing microorganisms, antibiotics should be discontinued);
3. Try to avoid using antibiotics as a preventive method.
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BufferPBS and DPBS are common washing buffer solutions. It is common to use PBS to wash the remaining complete culture medium before trypsin digestion, as the serum in the complete culture medium contains proteins that inhibit trypsin. If it is for some more difficult to digest cells, PBS without Ca and Mg ions must be used, as these ions also inhibit pancreatic enzyme activity. Purchase from legitimate suppliers to ensure quality, such as osmotic pressure, sterility, etc.PBS and DPBS are common washing buffer solutions. It is common to use PBS to wash the remaining complete culture medium before trypsin digestion, as the serum in the complete culture medium contains proteins that inhibit trypsin. If it is for some more difficult to digest cells, PBS without Ca and Mg ions must be used, as these ions also inhibit pancreatic enzyme activity.
Purchase from legitimate suppliers to ensure quality, such as osmotic pressure, sterility, etc. -
Cryopreservation solutionWhen thawing and reviving cells, it is not advisable to suspend them in the freezing tube for too long, and they should be transferred to the culture medium as soon as possible. If the condition of the cells is poor, the ratio of serum in the culture medium can be increased. The culture medium should be replaced the next day to replace the culture medium containing DMSO. It is also possible that some cells themselves have poor survival rate after freezing or have fewer cells during freezing. As noted above, it is necessary to understand whether there are any issues with the techniques used to freeze cells and whether there is a programmed cooling.When thawing and reviving cells, it is not advisable to suspend them in the freezing tube for too long, and they should be transferred to the culture medium as soon as possible.
If the condition of the cells is poor, the ratio of serum in the culture medium can be increased. The culture medium should be replaced the next day to replace the culture medium containing DMSO. It is also possible that some cells themselves have poor survival rate after freezing or have fewer cells during freezing. As noted above, it is necessary to understand whether there are any issues with the techniques used to freeze cells and whether there is a programmed cooling.Shanghai Xiaopeng can provide cell cryopreservation solution products:
1. Cell Cryopreservation Solution No. I (Cat. No. 3510/3511, phenol red-free)
2. Serum-Free Cell Cryopreservation Solution No. 2 (Cat. No. 3522/3523, phenol red-free)
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Pancreatic enzymes1.Once the digestion time gradually lengthens or the cells cannot digest, it is necessary to pay attention to whether pancreatic enzymes have been inactivated. Due to the fact that pancreatic enzymes belong to protein products, repeated freezing and thawing can easily affect their activity. The general method for preserving pancreatic enzymes is to pack 15 or 50 milliliters and freeze them at -20℃. Before use, a 37℃ water bath or incubator must be warmed up. After pancreatic enzyme digestion, cells should be washed with a buffer solution free of calcium and magnesium. 2. Poor quality pancreatic enzymes are also prone to being brought into mycoplasma or animal contamination sources. The pancreatic enzymes produced by VivaCell undergo radiation irradiation treatment to ensure quality and effectively prevent the introduction of mycoplasma. 3. Recombinant pancreatic enzymes can be used for clinical cultivation to avoid introducing unknown animal pathogens.1.Once the digestion time gradually lengthens or the cells cannot digest, it is necessary to pay attention to whether pancreatic enzymes have been inactivated. Due to the fact that pancreatic enzymes belong to protein products, repeated freezing and thawing can easily affect their activity. The general method for preserving pancreatic enzymes is to pack 15 or 50 milliliters and freeze them at -20℃. Before use, a 37℃ water bath or incubator must be warmed up. After pancreatic enzyme digestion, cells should be washed with a buffer solution free of calcium and magnesium.
2. Poor quality pancreatic enzymes are also prone to being brought into mycoplasma or animal contamination sources. The pancreatic enzymes produced by VivaCell undergo radiation irradiation treatment to ensure quality and effectively prevent the introduction of mycoplasma.
3. Recombinant pancreatic enzymes can be used for clinical cultivation to avoid introducing unknown animal pathogens.The general method for preserving pancreatic enzymes is to pack 15 or 50 milliliters and freeze them at -20℃. Before use, a 37℃ water bath or incubator must be warmed up. After pancreatic enzyme digestion, cells should be washed with a buffer solution free of calcium and magnesium.
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Medium -
SerumDue to different batch numbers, the serum components and nutrient content percentages may vary, and cells may need to adapt. Therefore, some batch numbers are suitable for nourishing nerve cells or mesenchymal stem cells, etc. When changing the serum batch number: 1. It is recommended to replace a small amount, with 1/3,1/2,2/3 gradual replacement; 2. Or increase the proportion of serum to increase nutrition, allowing cells to adapt more quickly; 3. Purchase sufficient serum at once to avoid frequent batch changes; 4. It is also important to pay attention to whether you have purchased counterfeit serum. Please be sure to purchase it from a reputable dealer. If you encounter excessively low prices and unclear channels, you need to be vigilant (not transported through the entire cold chain).Due to different batch numbers, the serum components and nutrient content percentages may vary, and cells may need to adapt. Therefore, some batch numbers are suitable for nourishing nerve cells or mesenchymal stem cells, etc.
When changing the serum batch number:
1. It is recommended to replace a small amount, with 1/3,1/2,2/3 gradual replacement;
2. Or increase the proportion of serum to increase nutrition, allowing cells to adapt more quickly;
3. Purchase sufficient serum at once to avoid frequent batch changes;
4. It is also important to pay attention to whether you have purchased counterfeit serum. Please be sure to purchase it from a reputable dealer. If you encounter excessively low prices and unclear channels, you need to be vigilant (not transported through the entire cold chain).Shanghai Xiaopeng provides:
All kinds of fetal bovine serum, specially processing technology Fetal Bovine Serum ,newborn bovine serum, adult bovine serum and other species of serum (pig, horse, sheep, immune), etc. -
Add a factorIn clinical and preclinical research, high-quality cell culture and differentiation tests often require the addition of cytokines. It is recommended to purchase highly active and high-purity recombinant proteins that do not contain animal and human derived substances to ensure that the product meets strict standards of activity and standardization. Commonly used additive factors, such as cytokines, are a class of small molecule proteins or peptides that can help cell growth, promote cell activation, directly participate in immune regulation and the formation of other cytokines, and serve as important bridges for message transmission between cells. Under normal physiological conditions, cells will naturally secrete after being stimulated, and different types of cytokines will also have different immune functions. However, excessive secretion of cytokines may also trigger inflammatory reactions, which can directly or indirectly induce or inhibit cell apoptosis. This animal derived additive, MatrigelR, has a greater inter batch difference than fully synthesized or single component reagents. Care must be taken when using it.In clinical and preclinical research, high-quality cell culture and differentiation tests often require the addition of cytokines. It is recommended to purchase highly active and high-purity recombinant proteins that do not contain animal and human derived substances to ensure that the product meets strict standards of activity and standardization.
Commonly used additive factors, such as cytokines, are a class of small molecule proteins or peptides that can help cell growth, promote cell activation, directly participate in immune regulation and the formation of other cytokines, and serve as important bridges for message transmission between cells. Under normal physiological conditions, cells will naturally secrete after being stimulated, and different types of cytokines will also have different immune functions. However, excessive secretion of cytokines may also trigger inflammatory reactions, which can directly or indirectly induce or inhibit cell apoptosis.
This animal derived additive, MatrigelR, has a greater inter batch difference than fully synthesized or single component reagents. Care must be taken when using it.
Shanghai Xiaopeng provides:
Research-grade and clinical-grade cytokines, hormones, and small-molecule compounds. For inquiries, please consult your local authorized distributors or contact our Technical Support Department.
Vitrogel® and BioLamina® are both synthetic and component-immobilized reagents with more consistent and reproducible experimental results.
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illuminationThe ultraviolet rays in the light can damage Hepes, riboflavin, and tryptophan in the culture medium, forming hydrogen peroxide free radicals and causing toxic effects on cells.The ultraviolet rays in the light can damage Hepes, riboflavin, and tryptophan in the culture medium, forming hydrogen peroxide free radicals and causing toxic effects on cells.
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L-glutamineFor different cell cultures, a certain amount of glutamine needs to be added to the culture medium as an energy source for cultivating cells, participating in protein synthesis and nucleic acid metabolism. Glutamine deficiency can lead to poor cell growth and even death. Glutamine will degrade after a period of time in solution, and the degradation of glutamine leads to the formation of ammonia, which is toxic to some cells. When the culture medium containing glutamine is stored in a 4℃ refrigerator for more than 2 weeks, glutamine should be added again. Alternatively, glutamine culture medium with long-term effects can be selected.For different cell cultures, a certain amount of glutamine needs to be added to the culture medium as an energy source for cultivating cells, participating in protein synthesis and nucleic acid metabolism. Glutamine deficiency can lead to poor cell growth and even death. Glutamine will degrade after a period of time in solution, and the degradation of glutamine leads to the formation of ammonia, which is toxic to some cells.
When the culture medium containing glutamine is stored in a 4℃ refrigerator for more than 2 weeks, glutamine should be added again. Alternatively, glutamine culture medium with long-term effects can be selected.Shanghai Xiaopeng provides:
1. Stable Glutamine (L-Alanyl-L-Glutamine), Cat No.:C3547;
2. For all kinds of cell culture auxiliary reagents, please consult the responsible distributor or technical support department.
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Basal mediumDry powder culture medium: When self configuring, it is necessary to pay attention to whether additional nutrients or essential substances are added to the culture medium (e.g. sodium bicarbonate needs special attention) and adjust the pH. Liquid medium: Cells can still grow using incorrect/mismatched medium, but their characteristics may change. The commonly used pH indicator in culture media, phenol red, actually has a weak estrogenic effect, stimulating the proliferation of cells with estrogen receptors.
Dry powder culture medium: When self configuring, it is necessary to pay attention to whether additional nutrients or essential substances are added to the culture medium (e.g. sodium bicarbonate needs special attention) and adjust the pH.
Liquid medium: Cells can still grow using incorrect/mismatched medium, but their characteristics may change.
The commonly used pH indicator in culture media, phenol red, actually has a weak estrogenic effect, stimulating the proliferation of cells with estrogen receptors.Shanghai Xiaopeng provides:
A variety of serum-free media systems for different cells, as well as a variety of basal media. Please consult the responsible dealer or technical support department.
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Human environment and operation -
SterilizerAll instruments and equipment require regular maintenance and calibration to maintain normal performance.All instruments and equipment require regular maintenance and calibration to maintain normal performance.Do BD (Bowie Dick) tests regularly to ensure that the pressure, temperature and time meet the sterilization requirements.
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Microscope storage tableWhen observing cells under a microscope, the loading platform and tabletop of the cell culture dish or culture bottle must also be kept clean to avoid cross contamination.When observing cells under a microscope, the loading platform and tabletop of the cell culture dish or culture bottle must also be kept clean to avoid cross contamination.Shanghai Xiaopeng provides:
Pharmacidal, for Disinfecting Surfaces, Mycoplasma/Bacterial/Fungal Contamination Prevention, Cat No.: C3490. -
Water bathThe water bath is a device that can easily breed bacteria and mould in the cell room, During the process of warming, the culture bottle is prone to adhering to pollution sources and spreading everywhere, and can be brought into environments such as biosafety cabinets.The water bath is a device that can easily breed bacteria and mould in the cell room, During the process of warming, the culture bottle is prone to adhering to pollution sources and spreading everywhere, and can be brought into environments such as biosafety cabinets.
Sterile ultra pure water should be added to the water bath and antibacterial agents should be added Akouguard-2, Cat No.: C3481 can be selected to effectively disinfect any form of water bath environment.
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Biosafety cabinetThe biosafety cabinet should be kept clean and clear as much as possible, and non essential items should not be placed inside, as it can easily disrupt the airflow inside the operating cabinet. Before use, it is necessary to use a disinfectant solution (such as 75% alcohol or Pharmacinal) for wiping. After use, it is also necessary to disinfect and UV treat for more than 20 minutes. The high-efficiency filter (HEPA filter) of biosafety cabinets and ultra clean workbenches can easily become a source of pollution after breeding bacteria. A good operating environment can ensure the health of cells.The biosafety cabinet should be kept clean and clear as much as possible, and non essential items should not be placed inside, as it can easily disrupt the airflow inside the operating cabinet. Before use, it is necessary to use a disinfectant solution (such as 75% alcohol or Pharmacinal) for wiping. After use, it is also necessary to disinfect and UV treat for more than 20 minutes.
The high-efficiency filter (HEPA filter) of biosafety cabinets and ultra clean workbenches can easily become a source of pollution after breeding bacteria. A good operating environment can ensure the health of cells.Shanghai Xiaopeng provides:
1.Pharmacidal, for Disinfecting Surfaces, Mycoplasma/Bacterial/Fungal Contamination Prevention, Cat No.: C3490.
2.Regularly replace the biosafety cabinet and ultra clean workbench HEPA filter, detect wind speed and leakage.
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CO2 incubator1. The CO2 and temperature and humidity in the incubator need to be regularly calibrated, as these are factors closely related to cell growth; 2. The temperature and humidity of the incubator are very suitable for microbial growth. It is necessary to regularly disinfect the incubator chamber and stainless steel partition. The water used in the water tray needs to be sterilized and antibacterial agents added to prevent the growth of mold and fungi.
1. The CO2 and temperature and humidity in the incubator need to be regularly calibrated, as these are factors closely related to cell growth;
2. The temperature and humidity of the incubator are very suitable for microbial growth. It is necessary to regularly disinfect the incubator chamber and stainless steel partition. The water used in the water tray needs to be sterilized and antibacterial agents added to prevent the growth of mold and fungi.
Shanghai Xiaopeng provides:
1. Pharmacidal, for Disinfecting Surfaces, Mycoplasma/Bacterial/Fungal Contamination Prevention, Cat No.: C3490;
2. Akouguard-1, Cat No.: C3480, effectively disinfects the water tray in the incubator;
3. Invest in a sterilizable CO2 incubator and sterilize it regularly.
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Cellulara chamberUnpurified indoor air can detect 500 to 2000 planktonic bacteria per cubic meter. 2015 Pharmacopoeia Guidelines 9205 Guidelines for Microbial Monitoring and Control in Drug Cleanliness Laboratories, which specify the quantity requirements for planktonic and settling bacteria of different cleanliness levels. Slippers or sandals should never be worn inside the cell room to avoid the introduction of foot mold. You should wear fully wrapped shoe covers or specialized shoes.Unpurified indoor air can detect 500 to 2000 planktonic bacteria per cubic meter.
2015 Pharmacopoeia Guidelines 9205 Guidelines for Microbial Monitoring and Control in Drug Cleanliness Laboratories, which specify the quantity requirements for planktonic and settling bacteria of different cleanliness levels.
Slippers or sandals should never be worn inside the cell room to avoid the introduction of foot mold. You should wear fully wrapped shoe covers or specialized shoes.
1. According to the requirements, the cell room should be a closed, positive pressure, and clean room (with a cleanliness level of 1- 10). If limited by the current situation, medical mobile air purifiers can be considered to purify the air.
2. If there is concern about the leakage of pollutants, the cell room should also be designed for relative pressure.
3. Personnel entering and exiting the cell room should be controlled and trained to enter and operate to avoid polluting others.
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OperatorsThe average surface of the human body contains microorganisms ranging from 10e3-10e6 per/cm2 , and pollutants may be brought in by operators and clothing. When entering and exiting the cell room, it is necessary to wear suitable laboratory clothes to minimize the rise and fall of dust, dander, and accompanying microorganisms, in order to avoid pollution.
The average surface of the human body contains microorganisms ranging from 103-106 per/cm2 , and pollutants may be brought in by operators and clothing. When entering and exiting the cell room, it is necessary to wear suitable laboratory clothes to minimize the rise and fall of dust, dander, and accompanying microorganisms, in order to avoid pollution.
Human beings are carriers of bacteria, and entering the cell room should:
1. Frequent dressing: After six hours of wearing sterile and dustfree clothing, 1-6 microorganisms can be collected per square centimeter.
2. Speak less: There are 107 microorganisms per milliliter in saliva. You should wear a mask to operate.
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VibrationWhen cells are just passaged or resuscitated, shaking and shaking can cause delayed adhesion to the wall, and even aggregation into clusters.When cells are just passaged or resuscitated, shaking and shaking can cause delayed adhesion to the wall, and even aggregation into clusters.Avoid vibration or centrifugal equipment in or around the incubator.
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Cell -
Artificial selection/mutationWhen cells are cultured in vitro, they actually undergo continuous human selection, such as: 1. Pancreatic enzyme digestion is a repetitive process of human selection, in which easily digested cells are passaged; 2. Excessive differentiation and stimulation of cells may cause abnormal mutations. Although HeLa cells still retain human genetic sequences, abnormal mutations in chromosome numbers have been observed (the original HeLa cell had 46 chromosomes, and currently there are cases where 70-90 chromosomes have been observed); 3. The hES cell line has been confirmed to be more prone to mutations in the in situ oncogene (TP53) as the number of generations increases. References Human pluripotent stem cells recurrently acquire and expand dominant negative P53 mutations”- Florian T. Merkle et al., Nature volume 545, pages 229-233 (11 May 2017)
When cells are cultured in vitro, they actually undergo continuous human selection, such as:
1. Pancreatic enzyme digestion is a repetitive process of human selection, in which easily digested cells are passaged;
2. Excessive differentiation and stimulation of cells may cause abnormal mutations. Although HeLa cells still retain human genetic sequences, abnormal mutations in chromosome numbers have been observed (the original HeLa cell had 46 chromosomes, and currently there are cases where 70-90 chromosomes have been observed);
3. The hES cell line has been confirmed to be more prone to mutations in the in situ oncogene (TP53) as the number of generations increases.
References
Human pluripotent stem cells recurrently acquire and expand dominant negative P53 mutations”- Florian T. Merkle et al., Nature volume 545, pages 229-233 (11 May 2017)
In order to ensure the consistency of experimental materials, a Seed Stock and Working Stock should be established for long-term cell lines, and identity identification should be arranged regularly. -
The cell seeding density is too lowCell cell adhesion and communication occur between cells, participating in cell recognition, cell activation, and signal transduction. During proliferation, growth factors that promote growth are secreted, creating an environment conducive to growth. Once the density of passage is too low, both can cause slow proliferation or death.
Cell cell adhesion and communication occur between cells, participating in cell recognition, cell activation, and signal transduction. During proliferation, growth factors that promote growth are secreted, creating an environment conducive to growth. Once the density of passage is too low, both can cause slow proliferation or death.
When passaging cells, perform a sure cell count to ensure the density of the plate. -
AgingAs long as cells continue to grow and divide, aging will occur; Whether it is the primary cell or the immobilized cell line. Several characteristics of aging cells: reduced cell division (slower proliferation), abnormal increase in cell size, and even multinuclear phenomena ranging from four to eight nuclei can be observed under a microscope. If more such cells are found during the process of subculture, it indicates that the cells have aged. Aging cells do not immediately die, but rather remain in the cell population with passage, and apoptosis occurs after some time (up to a year or two). The gene expression profile of aging cells may change, and it is recommended to resuscitate from a seed bank or working bank.
As long as cells continue to grow and divide, aging will occur; Whether it is the primary cell or the immobilized cell line.
Several characteristics of aging cells: reduced cell division (slower proliferation), abnormal increase in cell size, and even multinuclear phenomena ranging from four to eight nuclei can be observed under a microscope. If more such cells are found during the process of subculture, it indicates that the cells have aged. Aging cells do not immediately die, but rather remain in the cell population with passage, and apoptosis occurs after some time (up to a year or two). The gene expression profile of aging cells may change, and it is recommended to resuscitate from a seed bank or working bank.
Shanghai Xiaopeng can provide Cell Senescence Detection, Cat no.: T80500001
The cell aging assay accurately determines the percentage of aged cells to determine whether to use cells with a lower number of passages to maintain consistency in the experimental material. -
Passage density too high (>90%)May cause changes in cell adhesion: Cells not only adhere to the matrix, but also have adhesion between cells. Once the fusion of cells is too high before passage, cells may experience aging. Even if some cells have too high fusion, they will stack up and cause aggregates instead of being monolayer cells. This change is often irreversible, and even if the cells are re laid for passage, they will never return to their original state (e.g. there are always gaps in culture, and they are stacked before they are fully grown).
May cause changes in cell adhesion: Cells not only adhere to the matrix, but also have adhesion between cells. Once the fusion of cells is too high before passage, cells may experience aging. Even if some cells have too high fusion, they will stack up and cause aggregates instead of being monolayer cells.
This change is often irreversible, and even if the cells are re laid for passage, they will never return to their original state (e.g. there are always gaps in culture, and they are stacked before they are fully grown).
Try to avoid too high fusion, and the fusion degree can be divided into plates when the degree of integration reaches about 80%.
Confluence: In cell culture, this refers to the proportion of cells on the culture surface (dish/bottle).
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Cell cryopreservation/thawingMaster the principle of "slow freezing and fast melting" and carry out programmed cooling. Slow freezing: Two methods: 1. Manual program cooling, place cells sequentially at 4 °C (30 minutes), -20 °C (1 hour),-80°C (overnight), and then transfer to liquid nitrogen for storage. 2. Program cooling box, the most widely used cooling method, is a specially designed freezing container (filled with isopropanol or relying on special metal heat conduction) that lowers cells to -80°C (overnight) at a rate of about -1C per minute, and then moves them to liquid nitrogen for storage. Quick melting: Quickly transfer the frozen cells stored in liquid nitrogen into a 37 °C preheated water bath (the water surface should be lower than the pipe mouth to avoid accidental entry of water into the frozen tube), so that the frozen cell suspension can quickly melt; This method can quickly pass through the cell freezing suspension. Over the temperature range of -20 ℃~0 °C for crystal formation, the ice crystals outside the cell rapidly melt during freezing, avoiding ice crystals from entering the cell to form recrystallization and causing secondary damage to the cell.
Master the principle of "slow freezing and fast melting" and carry out programmed cooling.
Slow freezing: Two methods:
1. Manual program cooling, place cells sequentially at 4 °C (30 minutes), -20 °C (1 hour),-80°C (overnight), and then transfer to liquid nitrogen for storage.
2. Program cooling box, the most widely used cooling method, is a specially designed freezing container (filled with isopropanol or relying on special metal heat conduction) that lowers cells to -80°C (overnight) at a rate of about -1C per minute, and then moves them to liquid nitrogen for storage.
Quick melting: Quickly transfer the frozen cells stored in liquid nitrogen into a 37 °C preheated water bath (the water surface should be lower than the pipe mouth to avoid accidental entry of water into the frozen tube), so that the frozen cell suspension can quickly melt; This method can quickly pass through the cell freezing suspension.
Over the temperature range of -20 ℃~0 °C for crystal formation, the ice crystals outside the cell rapidly melt during freezing, avoiding ice crystals from entering the cell to form recrystallization and causing secondary damage to the cell.
Shanghai Xiaopeng provides cell cryopreservation solution products:
1. Cell Cryopreservation Solution No. I (Cat. No. 3510/3511, phenol red-free)
2. Serum-Free Cell Cryopreservation Solution No. 2 (Cat. No. 3522/3523, phenol red-free)
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Microbial contamination -
VirusCPE (cytopathic effect) can be easily observed in morphology, which may present as increased cell fragments, swelling, rounding, shedding, and large star suspension aggregation in the culture medium. In severe cases, local damaged plaques can be observed on the cell monolayer. The probability of virus contamination is relatively low and requires species pairing to proliferate.
CPE (cytopathic effect) can be easily observed in morphology, which may present as increased cell fragments, swelling, rounding, shedding, and large star suspension aggregation in the culture medium. In severe cases, local damaged plaques can be observed on the cell monolayer. The probability of virus contamination is relatively low and requires species pairing to proliferate.
Irradiated serum can be used to avoid the introduction of any live microorganisms into the culture system. -
Cross-contamination of cell linesCross-contamination between cell lines often occurs inadvertently, such as mixed contamination between different cell lines due to negligence in the experimental operation (when different cell lines share a bottle of medium, and the pipette tip and pipette are forgotten to be replaced), or the experimental operator miswrites the name of the cell line (Petri dish when subpassing, cryovial when freezing cells). Because this problem is so serious, most scientific journals have required that if you want to use cells for experiments, you must attach a "cell identification report" before you can publish an article.Cross-contamination between cell lines often occurs inadvertently, such as mixed contamination between different cell lines due to negligence in the experimental operation (when different cell lines share a bottle of medium, and the pipette tip and pipette are forgotten to be replaced), or the experimental operator miswrites the name of the cell line (Petri dish when subpassing, cryovial when freezing cells). Because this problem is so serious, most scientific journals have required that if you want to use cells for experiments, you must attach a "cell identification report" before you can publish an article.
Shanghai Xiaopeng provides:
1. STR Authentication of Human Cell Line, Cat No.: T802000011.
STR identification is required in the following cases:1). Establishment of new cell lines in the laboratory 2). Cells are derived from tissue-primary cells 3). Cells are not functioning properly 4). Publication 5). Routine periodic appraisal;
2. Species ldentification - identification of 15 common model animals, Cat No.: T80300001.
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MycoplasmaWhen contaminated with mycoplasma, the cell culture medium may still be clear or slightly yellow. Mycoplasma is an extremely common pollutant in cell culture experiments, with a probability of occurrence greater than 50%.Mycoplasma is commonly found in the human skin, respiratory tract, reproductive tract, and digestive tract. Incomplete disinfection of instruments can also easily introduce Mycoplasma into the culture system. Due to their very small size (0.15-0.3um) and relatively slow growth rate compared to bacteria, it is not easy to detect contamination using conventional methods.Mycoplasma can comprehensively affect cell morphology, proliferation, gene expression, protein synthesis, and metabolic reactions. At the same time, due to the fact that up to 30-50% of the DNA/RNA and proteins mentioned can be sources of Mycoplasma, there may be deviations in the results of QPCR or Western Blot experiments.
When contaminated with mycoplasma, the cell culture medium may still be clear or slightly yellow. Mycoplasma is an extremely common pollutant in cell culture experiments, with a probability of occurrence greater than 50%.Mycoplasma is commonly found in the human skin, respiratory tract, reproductive tract, and digestive tract. Incomplete disinfection of instruments can also easily introduce Mycoplasma into the culture system. Due to their very small size (0.15-0.3um) and relatively slow growth rate compared to bacteria, it is not easy to detect contamination using conventional methods.Mycoplasma can comprehensively affect cell morphology, proliferation, gene expression, protein synthesis, and metabolic reactions. At the same time, due to the fact that up to 30-50% of the DNA/RNA and proteins mentioned can be sources of Mycoplasma, there may be deviations in the results of QPCR or Western Blot experiments.
Shanghai Xiaopeng offers comprehensive solutions for Mycoplasma detection, treatment, and prevention:
1. MycoEZ, Mycoplasma qPCR Kit, Cat No.: M5010-0025;
2. Mycoplasma Removal Reagent：Protocol 1: MycoNator-1™ Antibiotic Solution 100X Conc., Cat No.: C3470+MycoNator-2™ Antibiotic Solution 100X Conc., Cat No.: C3471；
Protocol 2: MycoNator-3™ Antibiotic Solution 100X Conc. ,Cat No.: C3472；
3. Pharmacidal, for Disinfecting Surfaces, Cat No.: C3490;
4. Akouguard-1, Cat No.: C3480, Effectively disinfects water tray in CO₂ incubators.;
5. Akouguard-2, Cat No.:C3481, Effectively disinfects water baths of any type.
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MouldFloating debris resembling cotton flocs may be contaminated by fungi. This type of pollution does not cause the culture medium to turn yellow in the early stages, but mycelium can be observed under a microscope.
Floating debris resembling cotton flocs may be contaminated by fungi. This type of pollution does not cause the culture medium to turn yellow in the early stages, but mycelium can be observed under a microscope.
Shanghai Xiaopeng provides: Antibiotic Selection Protocol
1. C3420 (Penicillin-Streptomycin Solution)；
2. C3421 (Penicillin-Streptomycin Solution, 10X Conc.)；
3. C3422 (Penicillin-Streptomycin Nystatin Solution)；
4. C3423 (Penicillin-Streptomycin Amphotericin B Solution)；
5. C3424 (Penicillin-Streptomycin Neomycin Solution)；
6. C3430 (Amphotericin B Solution)；
7. C3440 (Nystatin Suspension)；
8. C3450 (Gentamycin Sulfate Solution)；
9. C3460 (Kanamycin Sulphate Solution).
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YeastA string of beads in the shape of an egg. The volume is 5-10 times smaller than that of cells. When pollution is severe, it can cause changes in the pH value of the culture medium, making it difficult to remove. When it undergoes budding and reproduction, it can be recognized from a large to small connected structure.
A string of beads in the shape of an egg. The volume is 5-10 times smaller than that of cells. When pollution is severe, it can cause changes in the pH value of the culture medium, making it difficult to remove. When it undergoes budding and reproduction, it can be recognized from a large to small connected structure.
Shanghai Xiaopeng provides: Antibiotic Selection Protocol
1. C3420 (Penicillin-Streptomycin Solution)；
2. C3421 (Penicillin-Streptomycin Solution, 10X Conc.)；
3. C3422 (Penicillin-Streptomycin Nystatin Solution)；
4. C3423 (Penicillin-Streptomycin Amphotericin B Solution)；
5. C3424 (Penicillin-Streptomycin Neomycin Solution)；
6. C3430 (Amphotericin B Solution)；
7. C3440 (Nystatin Suspension)；
8. C3450 (Gentamycin Sulfate Solution)；
9. C3460 (Kanamycin Sulphate Solution).
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BacteriaWhen bacterial contamination occurs, the cell culture medium may appear yellow/white cloudy.It may cause slow cell proliferation or changes in morphology (polygonal, multinuclear, etc.), resulting in vacuoles in the cytoplasm, non attachment of cells, abnormal pH changes observed in the culture medium, and a large number of black spots in the background observed under a microscope. The size, morphology, and movement of foreign objects can be used to determine whether bacterial contamination is present.
When bacterial contamination occurs, the cell culture medium may appear yellow/white cloudy.It may cause slow cell proliferation or changes in morphology (polygonal, multinuclear, etc.), resulting in vacuoles in the cytoplasm, non attachment of cells, abnormal pH changes observed in the culture medium, and a large number of black spots in the background observed under a microscope. The size, morphology, and movement of foreign objects can be used to determine whether bacterial contamination is present.
Shanghai Xiaopeng provides: Antibiotic Selection Protocol
1. C3420 (Penicillin-Streptomycin Solution)；
2. C3421 (Penicillin-Streptomycin Solution, 10X Conc.)；
3. C3422 (Penicillin-Streptomycin Nystatin Solution)；
4. C3423 (Penicillin-Streptomycin Amphotericin B Solution)；
5. C3424 (Penicillin-Streptomycin Neomycin Solution)；
6. C3430 (Amphotericin B Solution)；
7. C3440 (Nystatin Suspension)；
8. C3450 (Gentamycin Sulfate Solution)；
9. C3460 (Kanamycin Sulphate Solution).
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Swimming black dotsAt present, the scientific community has no consensus on the phenomenon of Swimming black dots, which should be a common term for microbial contamination caused by different species and genera.
Usually small black spots in the shape of egg circles or ball rods are observed, and active Brownian motion and obvious proliferation can be observed in the culture medium. They have cytotoxic effects and can cause cell growth to slow down or even lysis and apoptosis, and can appear in human and mammalian cell culture systems.If it can swim, it is usually some microbial contamination.
If it cannot swim, it may be formed by the precipitation of fetuin or carbonate apatite in the culture medium and serum.
References:
1.“Nanobacteria are Mineralo Fetuin Complexes”Didier Raoult, Patricia Renesto et al. PLoS Pathogens.2008 Feb.,4(2): e41
2.“Membrane Vesicles NucleateMineralo-organic Nanoparticles and induce Carbonate ApatitePrecipitation in Human Body Fluids”-Cheng-Yeu Wu, John D. Young et al. The Journal of BiolocalChemistry.2013 Oct.,288(42):30571-30584At present, the scientific community has no consensus on the phenomenon of Swimming black dots, which should be a common term for microbial contamination caused by different species and genera.
Usually small black spots in the shape of egg circles or ball rods are observed, and active Brownian motion and obvious proliferation can be observed in the culture medium. They have cytotoxic effects and can cause cell growth to slow down or even lysis and apoptosis, and can appear in human and mammalian cell culture systems.If it can swim, it is usually some microbial contamination.
If it cannot swim, it may be formed by the precipitation of fetuin or carbonate apatite in the culture medium and serum.
References:
1.“Nanobacteria are Mineralo Fetuin Complexes”Didier Raoult, Patricia Renesto et al. PLoS Pathogens.2008 Feb.,4(2): e41
2.“Membrane Vesicles NucleateMineralo-organic Nanoparticles and induce Carbonate ApatitePrecipitation in Human Body Fluids”-Cheng-Yeu Wu, John D. Young et al. The Journal of BiolocalChemistry.2013 Oct.,288(42):30571-30584
Shanghai Xiaopeng received samples from nearly 100 "Swimming black dots" from all over the China, and after analysis, 62% were bacteria and 38% were mycoplasma, includingspecies such as Mycoplasma hyorhinis (swine Mycoplasma), Mycoplasma bovis (bovine Mycoplasma), Actinobacterium ZXY023, Ralstonia sp./Ralstonia insidiosa, and Brevundimonas sp.
The introduction of "Swimming black dots"into cell cultures is likely attributed to substandard research reagents (e.g., trypsin, serum), contaminated consumables/environments, or improper handling by personnel.
Shanghai Xiaopeng can provide: Swimming black dots removal protocols:
Protocol 1: C3470 MycoNator-1 (MycoNator™-1 Antibiotic Solution 100X Conc.) and C3471 MycoNator-2 (MycoNator™-2 Antibiotic Solution 100X Conc.) and VivaCell Triple Antibiotic Solution.
Protocol 2: C3472 MycoNator-3 (mycoplasma treatment reagent 3) and VivaCell Triple Antibiotic Solution.
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