Cell culture tips(1)
i. Experimental supplies
1. Type of experimental products:
1.1. The cell culture laboratory supplies are all sterile, except for the glass container and pasteurpipet, all of which are sterile plastic products.
1.2. The TC level culture plate surface with coating polymer material to make cell adsorption, culture vessel types have Tflask, plates, dishes, rollerbottle etc., need to use in accordance with the experiment.
1.3. Plasticsterilepipet: 1 ml, 2 ml and 5 ml, 10 ml, 25 ml
1.4. Plastic centrifugal tube: 15ml, 50ml, all have 2 different materials, of which polypropylene(PP) is opaque material, polystyrene(PS) is transparent material, and the centrifuge tube suitable for materials can be selected according to the experimental needs.
1.5. Glasspastuerpipet: 9 inch to smoke ghost culture, etc.
1.6. Glass serum bottle (PyrexorDuranglassware) : 100ml,250ml,500ml,1000ml
2. Cleaning:
2.1. The newly purchased glass serum bottle will be soaked for a few hours at 0.1 to 0.05 NHCl, and then it will be used after washing.
2.2. Used glass serum bottle, sterilized with high pressure steam, cleaned after washing, and rinsed with secondary deionized water separately,
Do not wash with detergent.
3. The sterilization:
3.1. Experiment with glass bottles of serum to the aluminum foil paper cover cap and high pressure steam sterilization, 121 ℃, 15 lb, 20 minutes, in
Drying in oven.
3.2. Experiments with glass pasteurpipet at 170 ℃ dry sterilization, 4 hours.
3.3. The liquid or solid wastes available hypochloride 10% solution (hypochlorite, bleaching water) or steam
Pressure sterilization 121 ℃, 15 lb, 20 minutes.
ii. Medium
1. The liquid medium is stored at 4 ℃ refrigerator, avoid light, before the experiment in 37 ℃ warm in the sink.
2. Liquid culture medium (with serum) is stored for 6 months, during which glutamine may decompose, if cell growth is not good
You can add a little more of glutamine.
3. Preparation of powder culture medium (taking 1 liter for example) :
3.1. The cell culture medium usually requires 10% serum, so the composition of the powder culture medium is 900ml, and the pH is 7.2-7.4.
The NaHCO3 is added in addition. If the NaHCO3 powder is added directly to the liquid medium, the pH error will be caused, or the local alkaloid will be used.
And NaHCO3 powder powder medium should be dissolved after mixing respectively, and then use CO2 gas to adjust pH, rather than using acid (HCl) or strong alkali (NaOH), because of chloride ions may contribute to cell growth, and storage medium pH is easy to change.
Biological 3.2. Materials: pure water (milli-Q water or secondary to three times distilled water, water quality is very important), powder medium, NaHCO3
Electromagnetic stirrer, sterile serum bottle, 0.1 or 0.2mm sterile filtration membrane, pH meter, vacuum pump, CO2 gas.
3.3. Steps:
3.3.1. The powder medium was dissolved in the 700mln-q water and stirred to dissolve it.
3.3.2. The appropriate amount of NaHCO3 powder was dissolved in the water of 200mlmilli-Q water, stirred and dissolved, and then passed into the CO2 gas until it was saturated, about 3-5 minutes.
3.3.3. Mix the dissolved and saturated CO2 with the NaHCO3 solution into the dissolved liquid medium.
The pH of the mixing solution should be 7.2-7.4, unless the pH value is too large, it is not necessary to adjust the pH.
If it is too basic, it can be added to the CO2 gas to adjust the pH.
The pH increases by 0.1 to 0.2 when the medium is filtered by a vacuum pump.
3.3.4. 0.1 or 0.1 mm aseptic filtration membrane filtration sterilization, packing to sterile container at the same time, culture medium type, date, number, stored at 4 ℃.
(the serum can also be added to the medium to filter)
3.3.5. The preparation of the culture medium shall be tested for growth and contamination.
iii. Antibiotics
1. The cell culture medium of the cell is not antibiotic
1.1. Cultivate the cell lines introduced by ATCC, without antibiotics in the medium.
1.2. Cultivate the cell lines introduced from other laboratories, and make token-freeze medium to be added to the culture. After the tokenfreeze has been tested through contamination, antibiotics are not added to the mass culture.
2. When sending live cells, the medium must be filled with the whole flask, and antibiotics (penicillin 100units/ml+ streptomycin 100ug/ml) should be added.
3. If you want to detect mycoplasma, do not add gentamycin in the culture medium, because gentamycin will inhibit the growth of mycoplasma.
4. Remove the bacteria pollution of antibiotic mixed formula: penicillin 250 units/ml, streptomycin 250 ug/ml, neomycin250ug/ml, 2.5 units/ml, bacitracin note after mixing drug toxicity will be enhanced.
iv. serum
1. Must be stored in serum ~ 20-70 ℃, if stored at 4 ℃, no more than a month.
If one does not run out of a bottle of, can be 40 ~ 45 ml packing in a sterile 50 ml centrifuge tube, because the serum frozen volume will increase by about 10%, this expansion volume of the space must be reserved, pollution or containers of frost crack happens easily otherwise.
2. The serum provided by the general manufacturer is sterile and does not need to be sterilized.
If the serum is found to have a number of suspended objects, the serum can be added to the medium to filter the serum together.
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3.bottles (500 ml) serum thawing steps (to defrost method) : - 20 ℃ or 70 ℃ to 4 ℃ refrigerator dissolve one day, to the whole solution at room temperature before repackaging, general with 50 ml sterile centrifuge tube can be separated into 40 ~ 45 ml different.
In the process of dissolution, it is necessary to shake uniformly (not to create bubbles), to make the temperature and composition equal to one, to reduce the occurrence of shen dian.
Don't directly from 20 ℃ to 37 ℃ directly thawing, due to the temperature change is too big, easy to cause protein precipitation and precipitates.
4. Heat inactivated refers to 56 ℃, 30 minutes to heat fully thawed in the serum.
The rules must be uniformly shaken during heating.
The purpose of this heat treatment is to activate the complement of the serum.
Unless necessary, this heat treatment is generally not recommended because of the significant increase in the sediment and the quality of the serum.
The complement was destroyed.
5. Don't put the serum at 37 ℃ for too long, if placed at 37 ℃ for too long, serum becomes cloudy, and the composition of many less stable in serum are so damaged, and affect the quality of serum.
6. Serum deposits
6.1. The floe objects: occurrence of many kinds of reasons, but a common reason is that in the serum lipoprotein (lipoprotein) degeneration and thawed in serum fibrin (fibrin), caused by the blood of the floe precipitates content will not affect the quality of the serum itself.
To reduce these coagulant deposits, the centrifuge 3000rpm,5min removal, or centrifugation can be added to the medium to filter together.
It is not recommended to remove these coagulants with filtration steps, as they block the filtration membrane.
biological
6.2. The microscope , small black spots usually after heat treatment of serum, the formation of precipitates content increased significantly.
Some precipitates in microscope like , small black spots are often mistaken for serum suffered from pollution, and put the serum on to cultivate the microbial 37 ℃, but under the 37 ℃ environment, and makes the precipitates content increased, more will be mistaken for microbial proliferation, but to cultivate bacteria detection of medium, and no pollution.
In general, this small black spot should not affect cell growth, but if the serum is suspected, it should be stopped immediately and replaced with another batch of serum.