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, the test article was taken out of the workbench and the aseptic operation was 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
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 is coated with high molecular substances to allow the cells to adsorb. The culture vessels are Tflask, plates, dishes, roller bottles, etc., which are used according to the experiment.
1.3. plastic sterile pipet: 1 ml, 2 ml, 5 ml, 10 ml, 25 ml
1.4. Plastic centrifuge tube: 15 ml, 50 ml, available in 2 different materials, 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: 100 ml, 250 ml, 500 ml, 1000 ml
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 oC, 15 lb, 20 minutes, and dried in oven.
3.2. Experiment with glass pasteur pipet Sterilize at 170 oC 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 in a 4 oC freezer to avoid light and warm it in a 37 oC tank 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 medium, Table 1). Dissolve in 200ml of milli-Q water, stir to dissolve, and 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 deviation 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, H 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 oC. (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.
antibiotic
1. Cell culture medium of cell bank without antibiotics
1.1. Culture the cell line introduced from ATCC without antibiotics in the medium.
1.2. Incubate cell lines introduced from other laboratories, and add antibiotics to the medium before making the token freeze. After the token freeze passes the pollution test, no antibiotics are added when the culture is large.
2. When sending live cells, the antibiotics (penicillin 100 units/ml + streptomycin 100 ug/ml) must be added when the culture fluid is to be filled with the entire flask.
3. To detect mycoplasma, gentamicin should not be added to the medium, as gentamicin will inhibit the growth of mycoplasma.
4. Antibiotic mixed formula for removing bacterial contamination: penicillin 250 units/ml, streptomycin 250 ug/ml, neomycin 250 ug/ml, bacitracin 2.5 units/ml, note that the toxicity of the drug will increase after mixing.
5. Types and concentrations of antibiotics used: working concentration. Storage temperature. Kill bacteria
Penicillin 100 units/ml -20°C G(+)
Streptomycin 100 ug/ml -20°C G(+) and G(-) bacteria
Chlotetracycline 50 ug/ml -20°C G(+) and G(-) bacteria
Gentamicin 50 ug/ml -20°C G(+) and G(-) bacteria, mycoplasma
Amphotericin B 2.5 ug/ml -20°C yeast and molds
Nystatin 50 ug/ml -20°C yeast and molds
Fungizone 2.5ug/ml -20°C yeast and molds
serum
1. Serum must be stored at –20 to -70 oC. Do not exceed one month if stored at 4 oC. If one bottle cannot be used at a time, 40 to 45 ml can be dispensed into a sterile 50 ml centrifuge tube. Since the volume of the serum will increase by about 10% when frozen, the volume of the expansion volume must be reserved, otherwise contamination may occur. Or the case where the container is cracked.
2. The serum supplied by the general manufacturer is sterile and does not require sterile filtration. If serum is found to have many suspensions, the serum can be added to the medium for filtration and the serum should not be filtered directly.
3. Bottled (500ml) serum thawing step (gradual thawing method): -20 oC or –70 oC to 4 oC. Dissolve in the refrigerator for one day, then dissolve at room temperature and then dispense. Usually use 50 ml sterile centrifuge tube to dispense. 40 to 45 ml. During the dissolution process, it should be shaken regularly (be careful not to cause bubbles), so that the temperature and composition are uniform, and the precipitation is reduced. Do not thaw directly from –20 oC to 37 oC. Because the temperature changes too much, it is easy to cause protein to coagulate and precipitate.
4. heat-inactivation refers to serum that has been completely thawed at 56 oC for 30 minutes. Regularly shake evenly during heating. The purpose of this heat treatment is to deactivate the complement in the serum. This heat treatment is generally not recommended unless necessary, as it causes a significant increase in sediment and affects the quality of the serum. The reactions involved in complement participation are: cytolytic activities, contraction of smooth muscle, release of histamine from mast cells and platelets, enhanced phagocytosis, chemotaxis and activation of lymphocytic and macrophage cell type.
5. Do not leave the serum at 37 oC for too long. If it is left at 37 oC for too long, the serum will become cloudy, and many of the more unstable components in the serum will be destroyed, which will affect the quality of the serum.
6. Serum deposits
6.1. Flocs: There are many reasons for this, but the common cause is the degeneration of lipoprotein in serum and fibrin in the serum after thawing. These flocculation deposits do not Affect the quality of serum itself. If you want to reduce these flocculation, you can remove it by centrifugation at 3000 rpm for 5 min, or after centrifugation, the supernatant can be added to the medium for filtration. It is not recommended to remove these flocculation deposits by a filtration step as it will block the filter membrane.
6.2. “Small black spots†observed under the microscope: The serum that is usually heat treated will have a significant increase in the formation of precipitates. Some sediments are observed under the microscope as "small black spots", which are often mistaken for contamination of the serum, and the serum is placed at 37 oC to cultivate the "microorganism", but at 37 oC, it will cause this to sink. The increase in the number of deposits may be mistaken for the proliferation of microorganisms, but it is detected by the medium in which the bacteria are cultured, and there is no pollution. In general, this small black spot should not affect the growth of the cells, but if the quality of the serum is suspected, it should be immediately stopped and another batch of serum should be replaced.
7. Serum growth test
7.1. Materials:
7.1.1. MDCK cell (ATCC CCL-34 or CCRC 60004)
7.1.2. a-MEM (alpha modified minimal essential medium, GibcoBRL 12000-022)
7.1.3. 6-well TC plate (or 35mm TC dish)
7.1.4. methanol
7.1.5. glacial acetic acid
7.1.6. 10 % Giemsa solution (GibcoBRL 10092-013)
7.2. Steps:
7.2.1. Culture MDCK cells in T75 with a-MEM with 10% FBS (tested) to 80% confluency.
7.2.2. Treat cells with trypsin-EDTA, centrifuge, add appropriate amount of serum-free a-MEM to make a cell suspension, and measure the cell concentration. The cell concentration was diluted 1 x 102 viable cells/ml with serum-free a-MEM.
7.2.3. Inoculate 1 ml of cell suspension into a 6-well plate and add 1 ml of a-containing different concentrations of serum (20%, 10%, 4%, 2%, 1%, 0.4%). MEM, the final concentration of serum is 10%, 5%, 2%, 1%, 0.5%, 0.2%. The control group was tested simultaneously with the tested serum.
7.2.4. 37 oC, 5 % CO2 incubator for 5-7 days, no need to change the medium during the period, until the cell community is large enough to be observed by the naked eye, and the communities do not touch each other.
7.2.5. 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.
7.2.6. Remove the fixative and wash twice with water.
7.2.7. Add 1 ml of 10% Giemsa solution and let stand for 2-3 min at room temperature.
7.2.8. Remove the dye solution and wash it twice.
7.2.9. Counting the number of communities by the naked eye
7.2.10. Calculate SPE ( Serum Plating Efficiency ): SPE = ( no. of colonies / well ) / 100 x 100 %
7.2.11. Calculate RPE (Relative Plating Efficiency): SPE = [ total colonies of six well (test) / Total colonies of six well (control) ] x100 %
7.3. Comparing the RPE of each concentration of serum medium, the effect of the serum to be tested on cell growth can be known.
7.4. Order a large number of good sera from the same lot and store at –70 oC
Frozen cell activation
1. The principle of activation of frozen cells is rapid thawing to avoid re-crystallization of ice crystals and cause damage to cells, leading to cell death.
2. After cell activation, it takes about a few days, or one to two generations of the cell, its cell growth or characteristic performance will return to normal (such as the production of monoclonal antibodies or other proteins).
3. Materials
3.1 37 oC constant temperature water tank
3.2 Fresh medium
3.3 sterile pipette / centrifuge tube / flask
3.4 Liquid nitrogen or dry ice container
4. Steps:
4.1 Operators should wear protective masks and gloves to prevent possible damage to the freezing tube.
4.2 Remove the cryotube from the liquid nitrogen or dry ice container and check if the lid is tight. Due to the thermal expansion and contraction process, the lid is easy to loosen.
4.3 Place the fresh medium in a 37 °C water bath and return to warm. After returning to temperature, spray 70% alcohol and wipe it, and transfer it to the aseptic table.
4.4 Remove the cryotube and immediately thaw it in a 37 °C water bath. Gently shake the cryotube to melt it in 1 minute. Wipe the outside of the storage tube with 70% ethanol and transfer it to the aseptic table.
4.5 Remove 0.9 ml of the thawed cell suspension, slowly add the medium in the culture vessel (diluted 1:10 to 1:15), mix well, and incubate in a CO2 incubator. Another 0.1 ml of the thawed cell suspension was taken for survival testing.
4.6 Immediately after thawing, the cryoprotectant (such as DMSO or glycerol) is removed, depending on the cell type. In general, most of the cryoprotectant is not required to be removed immediately. However, if it is to be removed immediately, add the thawed cell suspension to a centrifuge tube containing 5-10 ml of medium, centrifuge at 1,000 rpm for 5 minutes, remove the supernatant, add fresh medium, mix well, and add to CO2. Box culture.
4.7 If the cryopreservative is not removed immediately, the medium is changed every other day after thawing.
Cell subculture
1. When cells are grown to high density, they must be colonized into new culture flasks. The typical dilution ratio is 1:3 to 1:6, depending on the cell type.
2. Material:
2.1. Dulbecco's phosphate-buffered saline, Ca++/Mg++ free, D-PBS, GibcoBRL 21600-010
2.2. trypsin-EDTA solution (0.05% trypsin-0.53mM EDTA-4Na, GibcoBRL 25300-062): Dispense in 10 ml in a 15 ml sterile centrifuge tube, store at –20 oC, and place in a 37 oC sink before use. Back to temperature.
2.3. Fresh medium
2.4. Sterile straws / centrifuge tubes / culture flasks
3. Steps:
3.1. adherent cells (adherent cells)
3.1.1. Aspirate the old culture solution.
3.1.2. Wash cells one or two times with D-PBS.
3.1.3. Add trypsin-EDTA solution (1ml/25cm2, 2ml/75cm2) for 37 minutes at 37 oC and observe under inverted microscope. When the cells are to be separated and appear round granular, trypsin-EDTA solution is aspirated. (If trypsin-EDTA is not removed, trypsin-EDTA is added, and the appropriate amount of serum-containing fresh medium is added to terminate trypsin. After centrifugation, the supernatant is aspirated.)
3.1.4. Tap the culture flask to remove the cells from the bottle wall, add appropriate amount of fresh medium, and pipette up and down several times to break up the cell mass. After mixing evenly, transfer to a new culture bottle according to the dilution ratio. , cultured under normal culture conditions.
3.2. Suspension cells
3.2.1. Aspirate the cell culture medium, place in a centrifuge tube, and centrifuge at 1000 rpm for 5 minutes.
3.2.2. Aspirate the supernatant, add an appropriate amount of fresh medium, mix well, transfer to a new flask according to the dilution ratio, and culture under normal culture conditions.
3.3. Hybridoma (hybridoma)
3.3.1. Some hybridoma cells need to be cultured for more than three days to produce antibodies. If the medium is changed, antibodies may be lost. Therefore, subculture can be changed without changing the medium after centrifugation, and the cell concentration can be diluted by directly adding fresh medium. If the volume is too large, it can be placed obliquely or colonized into a new culture flask.
Cell Culture Medium Technical Overview
Aseptic operation basic technology
1. Before the experiment, the sterile room and the laminar flow were sterilized by UV lamp for 30-60 minutes, the aseptic operation was carried out with 70 ethanol, and the aseptic console fan was turned on for 10 minutes. Only begin the experimental operation. Only one cell line was treated in the second operation, and the medium was not shared even if the medium was the same to avoid misunderstanding or cell contamination. After the experiment, the test article was taken out of the workbench and the aseptic operation was 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. Aseptically operated work area should be kept clean and spacious, necessary items such as test tube racks, pipettes or pipette boxes, etc.
For temporary placement, 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. Care should be taken with cell lines from human or viral infections and an appropriate level of aseptic table (at least Class II). 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
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 is coated with high molecular substances to allow the cells to adsorb. The culture vessels are Tflask, plates, dishes, roller bottles, etc., which are used according to the experiment.
1.3. plastic sterile pipet: 1 ml, 2 ml, 5 ml, 10 ml, 25 ml
1.4. Plastic centrifuge tube: 15 ml, 50 ml, available in 2 different materials, 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: 100 ml, 250 ml, 500 ml, 1000 ml
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 oC, 15 lb, 20 minutes, and dried in oven.
3.2. Experiment with glass pasteur pipet Sterilize at 170 oC 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 in a 4 oC freezer to avoid light and warm it in a 37 oC tank 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 medium, Table 1). Dissolve in 200ml of milli-Q water, stir to dissolve, and 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 deviation 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 oC. (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.
antibiotic
1. Cell culture medium of cell bank without antibiotics
1.1. Culture the cell line introduced from ATCC without antibiotics in the medium.
1.2. Incubate cell lines introduced from other laboratories, and add antibiotics to the medium before making the token freeze. After the token freeze passes the pollution test, no antibiotics are added when the culture is large.
2. When sending live cells, the antibiotics (penicillin 100 units/ml + streptomycin 100 ug/ml) must be added when the culture fluid is to be filled with the entire flask.
3. To detect mycoplasma, gentamicin should not be added to the medium, as gentamicin will inhibit the growth of mycoplasma.
4. Antibiotic mixed formula for removing bacterial contamination: penicillin 250 units/ml, streptomycin 250 ug/ml, neomycin 250 ug/ml, bacitracin 2.5 units/ml, note that the toxicity of the drug will increase after mixing.
5. Types and concentrations of antibiotics:
Working concentration. Storage temperature. Kill bacteria
Penicillin 100 units/ml -20°C G(+)
Streptomycin 100 ug/ml -20°C G(+) and G(-) bacteria
Chlotetracycline 50 ug/ml -20°C G(+) and G(-) bacteria
Gentamicin 50 ug/ml -20°C G(+) and G(-) bacteria, mycoplasma
Amphotericin B 2.5 ug/ml -20°C yeast and molds
Nystatin 50 ug/ml -20°C yeast and molds
Fungizone 2.5ug/ml -20°C yeast and molds
serum
1. Serum must be stored at –20 to -70 oC. Do not exceed one month if stored at 4 oC. If one bottle cannot be used at a time, 40 to 45 ml can be dispensed into a sterile 50 ml centrifuge tube. Since the volume of the serum will increase by about 10% when frozen, the volume of the expansion volume must be reserved, otherwise contamination may occur. Or the case where the container is cracked.
2. The serum supplied by the general manufacturer is sterile and does not require sterile filtration. If serum is found to have many suspensions, the serum can be added to the medium for filtration and the serum should not be filtered directly.
3. Bottled (500ml) serum thawing step (gradual thawing method): -20 oC or –70 oC to 4 oC. Dissolve in the refrigerator for one day, then dissolve at room temperature and then dispense. Usually use 50 ml sterile centrifuge tube to dispense. 40 to 45 ml. During the dissolution process, it should be shaken regularly (be careful not to cause bubbles), so that the temperature and composition are uniform, and the precipitation is reduced. Do not thaw directly from –20 oC to 37 oC. Because the temperature changes too much, it is easy to cause protein to coagulate and precipitate.
4. heat-inactivation refers to serum that has been completely thawed at 56 oC for 30 minutes. Regularly shake evenly during heating. The purpose of this heat treatment is to deactivate the complement in the serum. This heat treatment is generally not recommended unless necessary, as it causes a significant increase in sediment and affects the quality of the serum. The reactions involved in complement participation are: ytolytic activities, contraction of smooth muscle, release of histamine from mast cells and platelets, enhanced phagocytosis, chemotaxis and activation of lymphocytic and macrophage celltype.
5. Do not leave the serum at 37 oC for too long. If it is left at 37 oC for too long, the serum will become cloudy and many of the serum will be more turbid.
Unstable ingredients can also be destroyed, affecting the quality of serum.
6. Serum deposits
6.1. Flocs: There are many reasons for this, but the common cause is the degeneration of lipoprotein in serum and fibrin in the serum after thawing. These flocculation deposits do not Affect the quality of serum itself. If you want to reduce these flocculation, you can remove it by centrifugation at 3000 rpm for 5 min, or after centrifugation, the supernatant can be added to the medium for filtration. It is not recommended to remove these flocculation deposits by a filtration step as it will block the filter membrane.
6.2. “Small black spots†observed under the microscope: The serum that is usually heat treated will have a significant increase in the formation of precipitates. Some sediments are observed under the microscope as "small black spots", which are often mistaken for contamination of the serum, and the serum is placed at 37 oC to cultivate the "microorganism", but at 37 oC, it will cause this to sink. The increase in the number of deposits may be mistaken for the proliferation of microorganisms, but it is detected by the medium in which the bacteria are cultured, and there is no pollution. In general, this
Small black spots should not affect the growth of the cells, but if the quality of the serum is suspected, it should be stopped immediately and another batch of serum should be replaced.
7. Serum growth test
7.1. Materials:
7.1.1. MDCK cell (ATCC CCL-34 or CCRC 60004)
7.1.2. a-MEM (alpha modified minimal essential medium, GibcoBRL 12000-022)
7.1.3. 6-well TC plate (or 35mm TC dish)
7.1.4. methanol
7.1.5. glacial acetic acid
7.1.6. 10 % Giemsa solution (GibcoBRL 10092-013)
7.2. Steps:
7.2.1. Culture MDCK cells in T75 with a-MEM with 10% FBS (tested) to 80% confluency.
7.2.2. Treat cells with trypsin-EDTA, centrifuge, add appropriate amount of serum-free a-MEM to make a cell suspension, and measure the cell concentration. The cell concentration was diluted 1 x 102 viable cells/ml with serum-free a-MEM.
7.2.3. Inoculate 1 ml of cell suspension into a 6-well plate and add 1 ml of a-containing different concentrations of serum (20%, 10%, 4%, 2%, 1%, 0.4%). MEM, the final concentration of serum is 10%, 5%, 2%, 1%, 0.5%, 0.2%. The control group was tested simultaneously with the tested serum.
7.2.4. 37 oC, 5 % CO2 incubator for 5-7 days, no need to change the medium during the period, until the cell community is large enough to be observed by the naked eye, and the communities do not touch each other.
7.2.5. 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.
7.2.6. Remove the fixative and wash twice with water.
7.2.7. Add 1 ml of 10% Giemsa solution and let stand for 2-3 min at room temperature.
7.2.8. Remove the dye solution and wash it twice.
7.2.9. Counting the number of communities by the naked eye
7.2.10. Calculate SPE ( Serum Plating Efficiency ): SPE = ( no. of colonies / well ) / 100 x 100 %
7.2.11. Calculate RPE (Relative Plating Efficiency): SPE = [ total colonies of six well (test) / Total colonies of six well (control) ] x100 %
7.3. Comparing the RPE of each concentration of serum medium, the effect of the serum to be tested on cell growth can be known.
7.4. Order a large number of good sera from the same lot and store at –70 oC
Frozen cell activation
1. The principle of activation of frozen cells is rapid thawing to avoid re-crystallization of ice crystals and cause damage to cells, leading to cell death.
2. After cell activation, it takes about a few days, or one to two generations of the cell, its cell growth or characteristic performance will return to normal (such as the production of monoclonal antibodies or other proteins).
3. Materials
3.1 37 oC constant temperature water tank
3.2 Fresh medium
3.3 sterile pipette / centrifuge tube / flask
3.4 Liquid nitrogen or dry ice container
4. Steps:
4.1 Operators should wear protective masks and gloves to prevent possible damage to the freezing tube.
4.2 Remove the cryotube from the liquid nitrogen or dry ice container and check if the lid is tight. Due to the thermal expansion and contraction process, the lid is easy to loosen.
4.3 Place the fresh medium in a 37 °C water bath and return to warm. After returning to temperature, spray 70% alcohol and wipe it, and transfer it to the aseptic table.
4.4 Remove the cryotube and immediately thaw it in a 37 °C water bath. Gently shake the cryotube to melt it in 1 minute. Wipe the outside of the storage tube with 70% ethanol and transfer it to the aseptic table.
4.5 Take out 0.9 ml of the thawed cell suspension and slowly add it to the culture vessel (diluted by
1:10～1:15), mix well and put in CO2 incubator for cultivation. Another 0.1 ml of the thawed cell suspension was taken for survival testing.
4.6 Immediately after thawing, the cryoprotectant (such as DMSO or glycerol) is removed, depending on the cell type. In general, most of the cryoprotectant is not required to be removed immediately. However, if it is to be removed immediately, add the thawed cell suspension to a centrifuge tube containing 5-10 ml of medium, centrifuge at 1,000 rpm for 5 minutes, remove the supernatant, add fresh medium, mix well, and add to CO2. Box culture.
4.7 If the cryopreservative is not removed immediately, the medium is changed every other day after thawing.
Cell subculture
1. When cells are grown to high density, they must be colonized into new culture flasks. The typical dilution ratio is 1:3 to 1:6, depending on the cell type.
2. Material:
2.1. Dulbecco's phosphate-buffered saline, Ca++/Mg++ free, D-PBS, GibcoBRL 21600-010
2.2. trypsin-EDTA solution (0.05% trypsin-0.53mM EDTA-4Na, GibcoBRL 25300-062): Dispense in 10 ml in a 15 ml sterile centrifuge tube, store at –20 oC, and place in a 37 oC sink before use. Back to temperature.
2.3. Fresh medium
2.4. Sterile straws / centrifuge tubes / culture flasks
3. Steps:
3.1. adherent cells (adherent cells)
3.1.1. Aspirate the old culture solution.
3.1.2. Wash cells one or two times with D-PBS.
3.1.3. Add trypsin-EDTA solution (1ml/25cm2, 2ml/75cm2) for 37 minutes at 37 oC and observe under inverted microscope. When the cells are to be separated and appear round granular, trypsin-EDTA solution is aspirated. (If trypsin-EDTA is not removed, trypsin-EDTA is added, and the appropriate amount of serum-containing fresh medium is added to terminate trypsin. After centrifugation, the supernatant is aspirated.)
3.1.4. Tap the culture flask to remove the cells from the bottle wall, add appropriate amount of fresh medium, and pipette up and down several times to break up the cell mass. After mixing evenly, transfer to a new culture bottle according to the dilution ratio. , cultured under normal culture conditions.
3.2. Suspension cells
3.2.1. Aspirate the cell culture medium, place in a centrifuge tube, and centrifuge at 1000 rpm for 5 minutes.
3.2.2. Aspirate the supernatant, add an appropriate amount of fresh medium, mix well, transfer to a new flask according to the dilution ratio, and culture under normal culture conditions.
3.3. Hybridoma (hybridoma)
3.3.1. Some hybridoma cells need to be cultured for more than three days to produce antibodies. If the medium is changed, antibodies may be lost. Therefore, subculture can be changed without changing the medium after centrifugation, and the cell concentration can be diluted by directly adding fresh medium. If the volume is too large, it can be tilted or colonized into a new flask.
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