Cell culture knowledge (1)
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 tank, 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 culture flasks 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 serum use errors often cause cells to fail to survive.
5 What is FBS, FCS, CS, HS? FBS (fetal bovine serum) and FCS (fetal calf serum) are 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 adherence of adherent cells? How should the trypsin-EDTA concentration be 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. 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, and 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 centrifugation rate should be centrifuged?
To recover animal cells, the rate of centrifugation 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 ratio 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 contamination. To filter DMSO, a DMSO-resistant Nylon filter is required.
15 How to cryopreserve cells?
Cryopreservation method 1: Place the cryotube 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 excessive ice crystals, causing a large number of cell death. You can skip this step and put it directly into the -80 °C refrigerator, but the survival rate is slightly lower.
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. Except for highly experienced experts, most cell lines contaminated with mycoplasma cannot be distinguished by their 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 When detecting cell line contamination with mycoplasma, what should I do? Dispose of it directly after 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.
Cell culture knowledge (2)
Aseptic operation basic technology
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 is completed, the test article is taken out of the workbench and the aseptic operation is 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. A disinfectant (Zephrin 1:750) can be added to the sink to periodically change the water in the sink.
Laboratory supplies
1. Type?
1.1. Cell culture test supplies are sterile, except for glass containers and pasteur pipet, all of which are plastic sterile products.
1.2. The surface of the TC grade culture plate has coated polymer materials for cell adsorption. The culture vessels are Tflask, plates, dishes, roller bottles, etc., which are used according to the experiment.
1.3. plastic sterile pipet: 1ml, 2ml, 5ml, 10ml, 25ml
1.4. Plastic centrifuge tube: 15ml, 50ml, there are 2 different materials, of which polypropylene (PP) is opaque material, polystyrene (PS) is transparent material, and the centrifuge tube suitable for the material can be selected according to the experiment.
1.5. glass pastuer pipet: 9 inch, used to remove waste culture solution.
1.6. Pyrex or Duran glassware: 100ml, 250ml, 500ml, 1000ml
2. Cleaning? 2.1. The newly purchased glass serum bottle is first soaked in 0.1~0.05 N HCl for several hours before being washed. 2.2. Used glass serum bottles are sterilized by high pressure steam. After washing, rinse them with once and twice with deionized water. Do not use detergent to clean.
3. Sterilization?
3.1. Experimental glass bottles were covered with aluminum foil paper, autoclaved at 121 ° C, 15 lb, 20 minutes, and dried in oven.
3.2. Experimental glass pasteur pipet was sterilized by dry heat at 170 ° C for 4 hours.
3.3. Liquid or solid waste can be treated with 10% hypochloride solution (hypochlorous acid, ie bleach) or autoclaved at 121 ° C, 15 lb, 20 minutes.
Medium
1. Store the liquid medium in a refrigerator at 4 °C to avoid light. Warm it in a 37 °C water bath before the experiment.
2. Liquid medium (with serum) The storage period is six months, during which glutamine may decompose. If the cells are not growing well, an appropriate amount of glutamine may be added.
3. Powder medium preparation (in 1 liter as an example):
3.1. Cell culture medium usually requires 10% serum, so the volume of the powder medium is 900 ml and the pH is 7.2 - 7.4. NaHCO3 is added separately. If NaHCO3 powder is added directly to the liquid medium, it will cause pH error or local over-alkali. Therefore, the powder medium and NaHCO3 powder should be dissolved separately before mixing, and then the pH is adjusted with CO2 gas instead of strong acid (HCl) or strong alkali (NaOH), because chloride ions may have an effect on cell growth, and the medium is stored. The pH is subject to change. 3.2. Materials:
3.2.1. Pure water (milli-Q water or secondary to tertiary distilled water, water quality is very important)
3.2.2. Powder medium
3.2.3. NaHCO3 (Sigma S-4019)
3.2.4. Electromagnetic stirrer
3.2.5. Sterile serum bottles
3.2.6. 0.1 or 0.2 mm sterile filter membrane
3.2.7. pH meter
3.2.8. Vacuum pump
3.2.9. CO2 gas
3.3. Steps:
3.3.1. Dissolve the powder medium in 700 ml milli-Q water and stir to dissolve.
3.3.2. Weigh an appropriate amount of NaHCO3 powder (the amount varies depending on the type of medium, Table 1). Dissolve in 200ml milli-Q water, stir to dissolve, then pass CO2 gas to saturation for about 3-5 minutes.
3.3.3. Add the dissolved and saturated CO2 NaHCO3 solution to the dissolved liquid medium and mix. The pH of the mixed solution should be 7.2-7.4. Unless the pH value is too large, it is not necessary to adjust it with acid or alkali. If it is too alkaline, you can adjust the pH by introducing CO2 gas. When the medium passes through the filter membrane with a vacuum pump, the pH will increase by 0.1-0.2.
3.3.4. Filter and sterilize with a 0.1 or 0.2 mm sterile filter membrane, and dispense into a sterile container, label the medium type, date, bottle number, etc., and store at 4 °C. (Serum can also be added to the medium for filtration)
3.3.5. The prepared medium is prepared for growth and contamination testing.
4. Preparation of growth test for culture medium
4.1. Material:
4.1.1. MDCK cell (ATCC CCL-34 or CCRC 60004) 4.1.2. 6-well TC plate (or 35 mm TC dish)
4.1.3. methanol
4.1.4. glacial acetic acid
4.1.5. 10% Giemsa solution (GibcoBRL 10092-013)
4.2. Steps:
4.2.1. The MDCK cells were cultured in the medium to be tested, MDCK cells were seeded in a 6-well plate (or 35 mm TC dish), and each well was inoculated with 1 × 102 viable cells, and a control experiment was also performed.
4.2.2. After 5 to 7 days of inoculation, observe the growth of the cell population on a 100-fold inverted microscope until the cell population is large enough to be observed by the naked eye, and the communities do not touch each other. 4.2.3. Remove the medium and add 1 ml of Carnoy's fixative (methanol: glacial acetic acid? 3:1) and let stand for 10 min at room temperature.
4.2.4. Remove the fixative and wash it twice.
4.2.5. Add 1 ml of 10% Giemsa solution and let stand for 2-3 min at room temperature.
4.2.6. Remove the dye solution and wash it twice.
4.2.7. Count the number of colonies with the naked eye and compare them if the newly prepared or new batch of medium does not grow well for cells.
Cell culture knowledge (3)
How to choose a special cell line medium?
There is no fixed culture condition for culturing a certain type of cell. Cells cultured in MEM are likely to grow equally easily in DMEM or M199. In conclusion, MEM is the preferred method for adherent cell culture and RPMI-1640 for suspension cell culture is a good start. AIM V (12005) medium (SFM) is the best choice for serum-free culture for various purposes.
Is L-glutamine important in cell culture? Is it unstable in solution?
L-glutamine is important in cell culture. After the amino group is removed, L-glutamine can be used as a source of energy for cultured cells, involved in protein synthesis, and nucleic acid metabolism. L-Glutamine degrades over time in solution, but the exact rate of degradation has not been finalized. Degradation of L-glutamine leads to the formation of ammonia, which is toxic to some cells.
What is GlutaMAX-I? How is cultured cells using GlutaMAX-I? How stable is this dipeptide?
The GlutaMAX-I dipeptide is a derivative of L-glutamine whose unstable alpha-amino group is protected with L-alanine. A peptidase gradually cleaves the dipeptide and releases L-glutamine for use. The GlutaMAX-I dipeptide is very stable, and even after sterilization at 121 lbs for 20 minutes, the GlutaMAX-I dipeptide solution has minimal degradation, and under the same conditions, L-glutamine is almost completely degraded.
What medium can be omitted to add phenol red?
Phenol red is used as an indicator of pH in the medium: red in neutral, yellow in acidity, and purple in alkaline. Studies have shown that phenol red can mimic the effects of sterols (especially estrogens). In order to avoid sterol reaction, cultured cells, especially mammalian cells, are cultured without phenol red. Due to phenol red interference detection, some researchers did not use phenol red-containing medium for flow cytometry. Phenol red is used as an indicator of pH in the medium: red in neutral, yellow in acidity, and purple in alkaline. Studies have shown that phenol red can mimic the effects of sterols (especially estrogens). In order to avoid sterol reaction, cultured cells, especially mammalian cells, are cultured without phenol red. Due to phenol red interference detection, some researchers did not use phenol red-containing medium for flow cytometry. How to count live cells with trypan blue? Dilute the cell suspension to 200-2000 cells/ml with serum-free medium and add 0.1 ml of 0.4% trypan blue solution to 0.1 ml of cell suspension. Mix gently, and after a few minutes, count the cells using a hemocytometer. Living cells repel the trypan blue, so the cells stained blue are dead cells.
How to eliminate the pollution of tissue culture?
When important cultures are contaminated, researchers may try to eliminate or control pollution. First, determine that the contaminant is a bacterium, fungus, mycoplasma or yeast, isolate the contaminating cells from other cell lines, disinfect the culture vessel and the clean bench with a laboratory disinfectant, and inspect the HEPA filter. High concentrations of antibiotics and antimycotics may be toxic to some cell lines, and dose-response experiments are performed to determine the dose levels at which antibiotics and antimycotics produce toxicity. This is especially important when using antibiotics such as amphotericin B and antimycotics such as tylosin. The following are the recommended experimental procedures for determining toxicity levels and eliminating culture contamination. 1. Digest, count and dilute cells in antibiotic-free medium and dilute to the concentration of conventional cell passage. 2. Disperse the cell suspension into a multi-well plate or several small flasks. The selected antibiotic is added to each well over a concentration gradient. For example, amphotericin B recommends the following concentrations, 0.25, 0.50, 1.0, 2.0, 4.0, 8.0 mg/ml. 3. Observe cytotoxicity indicators every day, such as shedding, vacuoles, confluence and rounding. 4. After determining the level of antibiotic toxicity, culture the cells for 2 to 3 passages using a medium that is less than 2 to 3 times the concentration of the antibiotic. 5. Culture the cell generation in an antibiotic-free medium. 6. Repeat step 4. 7. Incubate for 4 to 6 passages in antibiotic-free medium to determine if contamination has been eliminated.
What is the role of sodium pyruvate in the medium?
Sodium pyruvate can be used as an alternative carbon source in cell culture. Although cells prefer glucose as a carbon source, cells can also metabolize sodium pyruvate without glucose.
Cell culture knowledge (4)
1. How to choose the medium?
There is no fixed culture condition for culturing a certain type of cell. Cells cultured in MEM are likely to grow equally easily in DMEM or M199. In conclusion, MEM is the preferred method for adherent cell culture and RPMI-1640 for suspension cell culture. The preferred medium for serum-free culture for various purposes is AIM V medium (SFM).
2. 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. Heat inactivated serum is recommended for immunological studies, culture of ES cells, insect cells, and smooth muscle cells.
3. Is L-glutamine important in cell culture?
Is it unstable in solution? L-glutamine is important in cell culture. After the amino group is removed, L-glutamine can be used as a source of energy for cultured cells, involved in protein synthesis, and nucleic acid metabolism. L-Glutamine degrades over time in solution, but the exact rate of degradation has not been determined. Degradation of L-glutamine leads to the formation of ammonia, which is toxic to some cells.
4. What is GlutaMAX-I?
How does the cultured cell utilize GlutaMAX-I? How stable is the dipeptide? The GlutaMAX-I dipeptide is a derivative of L-glutamine, and its unstable α-amino group is protected with L-alanine. A peptidase gradually cleaves the dipeptide and releases L-glutamine for use. The GlutaMAX-I dipeptide is very stable, and even after sterilization at 121 lbs for 20 minutes, the GlutaMAX-I dipeptide solution has minimal degradation, and under the same conditions, L-glutamine is almost completely degraded.
5. Why can I add phenol red to the medium?
Phenol red is used as an indicator of pH in the medium: red in neutral, yellow in acidity, and purple in alkaline. Studies have shown that phenol red can mimic the effects of sterols (especially estrogens). In order to avoid sterol reaction, cultured cells, especially mammalian cells, are cultured without phenol red. Due to phenol red interference detection, some researchers did not use phenol red-containing medium for flow cytometry.
6. How to count live cells with trypan blue?
The cell suspension was diluted to 200-2000 cells/ml with serum-free medium, and 0.1 ml of 0.4% trypan blue solution was added to 0.1 ml of cells. Mix gently, and after a few minutes, count the cells using a hemocytometer. Living cells repel the trypan blue, so the cells stained blue are dead cells.
7. How to eliminate the pollution of tissue culture?
When important cultures are contaminated, researchers may try to eliminate or control pollution. First, determine that the contaminant is a bacterium, fungus, mycoplasma or yeast, isolate the contaminating cells from other cell lines, disinfect the culture vessel and the clean bench with a laboratory disinfectant, and inspect the HEPA filter. High concentrations of antibiotics and antimycotics may be toxic to some cell lines, and dose-response experiments are performed to determine the dose levels at which antibiotics and antimycotics produce toxicity. This is especially important when using antibiotics such as amphotericin B and antimycotics such as tylosin. The following are the recommended experimental procedures for determining toxicity levels and eliminating culture contamination.
1) Digest, count and dilute cells in antibiotic-free medium and dilute to the concentration of conventional cell passage.
2) Disperse the cell suspension into a multi-well plate, or several small flasks. The selected antibiotic is added to each well over a concentration gradient. For example, amphotericin B recommends the following concentrations, 0.25, 0.50, 1.0, 2.0, 4.0, 8.0 mg/ml.
3) Observe cytotoxicity indicators every day, such as shedding, vacuoles, confluence and rounding.
4) After determining the antibiotic toxicity level, the cells are cultured for 2-3 passages using a culture medium having an antibiotic concentration of 2-3 times lower than the toxic concentration.
5) Culture the cells in a medium without antibiotics.
6) Repeat step 4.
7) Incubate for 4-6 generations in antibiotic-free medium to determine if contamination has been eliminated.
8. What is the role of sodium pyruvate in the medium?
Sodium pyruvate can be used as an alternative carbon source in cell culture. Although cells are more likely to use glucose as a carbon source, cells can also metabolize sodium pyruvate without glucose.
9. Hank's Balanced Salt Solution (HBS) is intended for use in air and does not require a CO2 incubator. what is the reason?
What are the essential functional differences between Hank's Balanced Salt Solution (HBS) and Earle's Balanced Salt Solution (EBS)? The main difference between HBS and EBS is the level of sodium bicarbonate, and the content of sodium bicarbonate in Eagles (2.2g/L). High in Hanks (0.35g/L). Sodium bicarbonate is equilibrated with a high level of CO2 to maintain the pH of the solution. In the air level of CO2 in the Eagles solution, the solution will become alkaline and the Hanks solution will become sour in the CO2 incubator. If you want to preserve tissue in a CO2 incubator, you need to use Eagles fluid. If you are just cleaning the tissue that will be stored in the cell culture medium, use Hanks' solution.
10. What is the difference between Qualified and Certified fetal bovine serum?
Certified fetal bovine serum includes all standard testing procedures performed by Qualified fetal bovine serum, and in addition to these standard tests, Certified fetal bovine serum has the following additional tests: End-Point Determination of Endotoxin Content Phage Test Biochemical Detection of Hormone Detection of hemoglobin detection Sf9 cell growth promotion and methodological detection
11. 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.
12. Does the method of preparing lipid-DNA affect transfection efficiency?
Yes. For LIPOFECTAMlNE Reagent, the dilution reagent was diluted in 100 μl of OPTI-MEM in 100 μl of OPTI-MEM. The two solutions were mixed and incubated for 15 minutes at room temperature. For LIPOFECTIN Reagent, allowing the diluted reagent and medium to incubate for at least 30 minutes prior to addition of the DNA solution can increase efficiency by a factor of three. Ensure that the complex is formed without serum. After incubation for 15 minutes, serum-containing medium (800 μl) was added to the complex. (Note that the above is the volume used for the 35mm culture dish). For LIPOFECTAMlNE Plus Reagent DNA should be first mixed with Plus Reagent prior to mixing with the liposomes. The procedure of LIPOFECTAMlNE 2000 allows DNA and liposomes to be mixed in a small volume, which can then be added directly without changing the medium.
13. When I used SF900 II, the cells grew well, but why is my protein product not as good as Graces and 10% fetal bovine serum?
If the protein of interest is a late protein, it will be expressed together with a protease that will act on the protein of interest. In serum-added media, these proteases act on proteins in the serum to keep the protein product of interest intact. In serum-free formulations, the only substrate for protease action is your protein of interest. To avoid this problem, add some protease inhibitors or add a small amount of serum (less than 1%) to allow the serum to provide a substrate for the protease.
14. How to detect endotoxin (heat source) levels?
The LAL (Limulus Amebocyte Lysate) test is the most sensitive and specific method for detecting bacterial endotoxin. Levin and Bang found that bacteria can cause intravascular agglutination of Limulus polyphemus. Endotoxin initiates an intracellular zymogen system (serine protease cascade) that, by modifying the lectin, produces a clear gel, a coagulated protein in the LAL, thereby forming an insoluble matrix. LAL reagent buffered sputum (blood cell) lysate. Endotoxin testing of fetal bovine serum was performed on Grand Island according to the Gel-clot method in the manual. Samples were diluted 1:10 with non-pyrogenic water prior to endotoxin testing of serum products. The diluted sample was boiled for 5 minutes to eliminate the inhibitor. (Usually, the presence of endotoxin-binding components in the blood inhibits the gel process, preventing endotoxin from reacting with LAL. Pre-heat treatment of the sample eliminates this inhibition.)
15. Can I use the Murashige Skog medium in solid form?
If a solid form of the medium is used, agar needs to be added. Sterilize before agar is added. Direct sterilization of the MS medium should be avoided. The agar solution should be autoclaved and the melted agar solution added to the MS medium.
16. Does the pH of the buffer prepared at 20 °C change at higher or lower temperatures?
For commonly used buffers, the pH varies with temperature. The table below shows the change in pH at a temperature change of 10 ° C. For example, PH7.4 Tris buffer is prepared at 20 ° C. The pH is 7.4-(2x0.310) = 6.7817 at 40 ° C. Prepared at room temperature (25 ° C) of
17. What is the pH of the Tris-HCl solution when used at 37 °C?
The pH of the buffer changes with temperature. The table below lists the different pH values ​​for 50 mM Tris-HC solutions at 4 ° C, 25 ° C, and 37 ° C.
18. What is the optimum pH and osmotic pressure for insect cell culture?
The pH of the growth medium has an effect on the proliferation of cells and the production of viruses or recombinant proteins. For most lepidopteran cell lines, most applications in the pH range of 6.0-6.4 work well. When culturing a lepidopteran insect cell line, the optimum osmotic pressure of the medium is 345-380 mOsm
. . . To ensure reliable and long-lasting cell culture methods, reduce technical problems and maintain pH and osmotic pressure within the ranges listed above.19. Does the High Five cell have any other names?
High Five cells are also known as Trichoplasia ni 5B1-4 and BTI-TN-5B1-4.
20. What is the concentration of detergent in High Five serum-free medium?
Concentration of detergent in High Five serum-free medium: 0.025 g/L Tween-80, 1.0 g/L Pluronic Poly-all.
21. What is the density of High Five cells frozen?
3.0x10E6 cells/ml
22. A white precipitate was found in my Drosophila medium and dissolved after heating. What is it? Is it harmful to my cells?
It may be glutamine precipitation, but is more likely to be L-tyrosine precipitation. The concentration of glutamine in the medium is 6 times higher than the typical 2 mM. The concentration of tyrosine is 25 times higher than in RPMI 1640 and is more difficult to dissolve than glutamine. Precipitation may also be a complex of more than one component. It may be caused by storage at a lower local temperature. As long as the precipitate can be dissolved under the culture conditions, there is no adverse effect on the experiment.
23. How to transfer sf9 cells from T25 bottles? Can it be digested with trypsin?
We strongly recommend the use of exfoliated cells because it is the least destructive and has the highest viability. The medium was allowed to flow on the cells by using a Pasteur pipette. As an option you can also tap the culture bottle gently. Trypsinize the cells only if absolutely necessary. Trypsinize a T25 bottle of sf9 cells: 1) Remove the medium. 2) Wash the cells with 2 ml of 1x PBS (sufficient to cover the cell surface) to remove PBS. 3) Add 2 ml of 1x trypsin EDTA (just cover the cell surface). 4) Incubate at 37 °C for 5 to 10 minutes. They were moving up after 5 minutes of detection under the instrument. 5) Add 2 ml of cell culture solution to the cells, transfer to a conical tube, wash the wall with 2 ml of culture medium, and transfer to the same conical tube. (FBS in the medium terminated the activity of trypsin.) 6) The cells were pelleted by centrifugation (1100 rpm). Remove the medium. 7) Resuspend the cells with the new medium. pass on. 24. What is the amount of heparin used in suspension culture of Sf9, Sf21, and high Five cells? To prevent the formation of cells in suspension culture, a heparin concentration of 10 units/ml cell suspension was used. 25. How to evaluate ES cell-qualified fetal bovine serum? Use D3 ES cells. This is a cell line that is very sensitive to growth promotion, growth inhibition and differentiation factors in fetal bovine serum. Correlation Growth Efficiency Analysis: The ability to initiate and support ES cell cloning was tested when ES cells were introduced into growth medium containing 10% fetal bovine serum at very low density. Cytotoxicity assay: The growth capacity of ES cells and feeder cells was measured when introduced into growth medium containing 30% fetal bovine serum at a very low density. Related Morphology and Differentiation Analysis: The ability of fetal bovine serum to support undifferentiated ES cell clones was examined. The degree of differentiation was assessed by alkaline phosphatase activity. Undifferentiated ES cells are dark reddish pink, with differentiated cells that are large, plump, and light in color. All analyses were performed without ESGRO, and the presence of ESGRO in the medium masked the problems caused by fetal bovine serum. (ESGRO or LIF is often used to keep ES cells in an undifferentiated state.) After culture, one out of about 8 batches can be used to culture ES cells.
26. How many generations can it pass before re-frozening sf9 cells? As the number of passages increases, will its infection capacity decrease?
Normally, cells should be returned to cryopreservation after 30 passages. Cell viability should be checked whenever you count. If more than 95% of the cells remain viable and double in about 30 hours, the cells can still be used. If vitality and doubling time decline, their appeal will not be effective.
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