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Note: Analytical grade chemicals must be used throughout. |
1) Phosphate buffered saline, pH 7.2 to 7.4 (PBS)
This is the simplest of basic salt solutions and is used for washing cells prior to cell disaggregation. PBS in the incomplete and complete form is available commercially. An incomplete solution of PBS contains no calcium or magnesium ions. A complete solution of PBS is used mainly in the preparation of specimen extracts and as diluent for viruses; the presence of calcium and magnesium ions stabilizes viruses, particularly poliovirus and other enteroviruses.
2) Solution A
NaC1 8.00 g
KC1 0.20 g
Na2HPO4 (anhydrous) 0.91 g
KH2 PO4 0.12 g
Dissolve the salts in 600–800 ml distilled H2O. Add 2 ml of 0.4% phenol red as pH indicator. Make up to 1000 ml with distilled H2O and autoclave at 10 psi (70 kPa) for 15 minutes (110°C). This gives a working solution of incomplete PBS (i.e. no calcium or magnesium ions present).
3) Solution B
MgC12.6H2O 0.10 g
Dissolve in 100 ml distilled H2O. Autoclave at 10 psi (70 kPa) for 15 minutes.
4) Solution C
CaC12 0.10 g
Dissolve in 100 ml distilled H2O. Autoclave at 10 psi (70 kPa) for 15 minutes.
5) Working solution of complete PBS
Add 1 part of Solution B and 1 part of Solution C to 8 parts of Solution A.
Alternatively use commercially prepared tablets or powder, following the manufacturer's instructions for reconstitution and sterilization.
6) Sodium bicarbonate solution
Together with gaseous CO2 this provides the buffering system for many cell culture media; it is also an essential metabolite.
NaHCO3 7.5 g
Dissolve in 50 ml distilled H2O and add 0.2 ml of 0.4% phenol red. Make up to 100 ml with distilled H2O, saturate with CO2 until orange in colour. Dispense in approximately 5 ml volumes in tightly-capped bottles. Autoclave at 10 psi (70 kPa) for 15 minutes.
7) Eagle's growth medium (GM) and Eagle's maintenance medium (MM)
Used for culturing RD and L20B cell lines in the presence of CO2:
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Growth medium |
Maintenance |
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Eagle's minimum essential medium (Earle's salts base, no bicarbonate) |
83.3 ml |
90.3 ml |
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L-glutamine 200 mM |
1.0 ml |
1.0 ml |
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Fetal calf serum |
10.0 ml |
2.0 ml |
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NaHCO3 solution 7.5% |
3.5 ml |
4.5 ml |
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HEPES 1M |
1.0 ml |
1.0 ml |
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Penicillin/streptomycin solutiona |
1.0 ml |
1.0 ml |
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0.4% phenol red |
0.2 ml |
0.2 ml |
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a Dissolve 1 x 106 units crystalline penicillin G and 1 g streptomycin sulphate in 100 ml PBS and sterilize by filtration; distribute into 5 ml volumes and store at -20°C. For use, add 1 ml of this stock solution to 100 ml medium to give a final concentration of 100 units penicillin and 100 μg streptomycin per ml.
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Good laboratory practice: Some manufacturers include supplementary reagents (such as HEPES and L-glutamine) with their basal media. Always check the manufacturer's formulations before adding further supplements. L-glutamine is unstable. Its half-life in medium at 4°C is about three weeks and at 36°C about one week. Aliquots of sterile L-glutamine stock solutions should be stored frozen at -20°C until use. |
8) Hank's growth medium (GM) and Hank's maintenance medium (MM)
Used for culturing RD and L20B cell lines in closed systems (tubes and flasks with tightened lids) without CO2. Standard incubators can be used.
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Growth |
Maintenance |
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Minimum essential medium (Hank's salts base, no bicarbonate) |
85.3 ml |
92.3 ml |
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L-glutamine 200 mM |
1.0 ml |
1.0 ml |
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Fetal calf serum |
10.0 ml |
2.0 ml |
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NaHCO3 solution 7.5% |
1.5 ml |
2.5 ml |
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HEPES 1M |
1.0 ml |
1.0 ml |
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Penicillin/streptomycin solutiona |
1.0 ml |
1.0 ml |
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0.4% phenol red |
0.2 ml |
0.2 ml |
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a Dissolve 1 x 106 units crystalline penicillin G and 1 g streptomycin sulphate in 100 ml PBS and sterilize by filtration; distribute into 5 ml volumes and store at -20°C. For use, add 1 ml of this stock solution to 100 ml medium to give a final concentration of 100 units penicillin and 100 μg streptomycin per ml. |
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9) Other antibiotics
Penicillin and streptomycin are the antibiotics most commonly used in cell and routine virus culture work; they are also the least expensive. Gentamicin is more expensive, but it is inhibitory to a wider range of bacteria and it is autoclavable. Gentamicin should be used at a final concentration of 50 μg/ml. Mycostatin may be used at 25 units/ml to counteract fungal and yeast contaminants; however, it is only fungistatic and not fungicidal, is rapidly inactivated at cell culture incubation temperature and some batches are slightly cytotoxic. Fungizone also often produces cytotoxic effects.
10) Cell dispersing agents
Trypsin and Versene (EDTA) are commonly used, either separately or combined. The proteolytic enzyme trypsin is particularly suitable for the digestion of cells from whole organs. It is also used for the removal of cells from glass or plastic, but the chelating agent Versene is probably as good. Solutions of trypsin and/or Versene should be prepared in PBS (incomplete) free of calcium and magnesium, as the presence of these ions increases the stability of the intercellular matrix thereby making detachment of the cells from the glass/plastic difficult.
Trypsin: Dissolve 1 g of Difco 1:250 trypsin in 400 ml PBS (without Ca, Mg) by gentle agitation with a magnetic stirrer for 30 minutes at 36°C. Membrane filter (pore size 0.22 μm), dispense into approximately 5 ml volumes in sealed bottles and store at -20°C.
Versene: (EDTA = disodium salt of ethylenediaminetetra-acetic acid). Dissolve 0.1 g of Versene in 10 ml distilled water. Distribute in 0.5 ml volumes in sealed bottles; autoclave 10 psi (70 kPa) for 15 minutes and store at room temperature. For use add 0.4 ml of this 1% solution to 20 ml PBS (without Ca, Mg) to give a final concentration of 0.02%.
11) Phenol red indicator
This indicator of pH is used in cell culture media. Prepare stock 0.4% w/v solution in distilled H2O. Distribute into sealed bottles; autoclave at 10 psi (70 kPa) for 15 minutes and store at room temperature. For media preparation, 1–2 ml of this solution per litre is usually adequate.