Eagle s medium

Eagle s media contain, in addition to the salts and glucose of BSS, 12 essential amino acids and 9 vitamins. Glutamine and, if required, antibiotics along with serum etc., have to be added also before use. 

The simpler forms of Eagle s media (and some other media) are available commercially in powdered form from for example Flow Laboratories or Gibco (see Appendix 3). This is the cheapest way of buying media if large quantities are used the powder occupies little space and may be stored in the cold room in sealed containers for 6-12 months. 

Eagle s media formulations (amino acid and vitamin components)... 

Preparation of Eagle s media from concentrated stock solutions... 

Approximately 2.5-5 X 10 cells were seeded into 24-well plates and allowed to adhere overnight. Dulbecco s modified Eagle s media (phenol red free) with supplements were added to the cells (Mitrovic et ah, 1994). The cells received no treatment, received interferon-y (IFN-y) (500 U/ml) and interleukin-1/8 (IL-1/3) (300 U/ml), or were pretreated with pentoxifylline (PTX) (1 mg/ml for 24 hr) before stimulation with IFN-y and IL-lj3. After 4-7 days of stimulation, cell supernatants were removed and stored at —40°C. Samples were thawed, and soluble TNF-Rl and TNF-R2 levels were assayed using enzyme-linked immunosorbent assay kits [human soluble tumor necrosis factor receptor type 1 (sTNF-Rl) and sTNF-R2 Quantikine kits, R D Systems, Minneapolis, MN]. For the sTNF-Rl kit the minimum detectable dose is 1 pg/ral. For the TNF-R2 kit the minimum detectable dose is 0.5 pg/ml. PTX was provided courtesy of Hoechst-Roussel Pharmaceuticals (Somerville, NJ). Values are expressed as means SD. 

Historically, the development of animal cell culture systems has been dependent upon the development of new types of tissue culture media. Mouse L cells and HeLa cells were developed using a balanced salt solution supplemented with blood plasma, an embryonic tissue extract, and/or serum. In 1955 Eagle developed a nutritionally defined medium, containing all of the essential amino acids, vitamins, cofactors, carbohydrates, salts, and small amounts of dialyzed serum (Table 1). He demonstrated that this minimal essential medium (MEM) supported the long-term growth of mouse L and HeLa ceils. Eagle s MEM was so well defined that the omission of a single essential nutrient eventually resulted in the death of these animal cells in culture. 

Basic animal cell culture media, such as Dulbecco s modified Eagle s medium, generally contain ... 

EROD activity is measured in the H411E cells as follows. The cells are seeded at 7,000 cells per well in 250 xL of Dulbecco s modified Eagles culture media (Tillitt et al., 1991). After an initial incubation period of 24 hours, the cells are dosed with 5 xL volumes of eiuiched SPMD extracts (cleanup of extracts generally includes dialysis and size exclusion chromatography [SEC]) and incubated for an additional 72 hours. Sample dose is typically expressed as g-equivalents triolein or whole SPMD per mg cellular protein. Multiple exposures are performed at each of six (typically) sample concentrations, using a dilution series. Afterwards, the microtiter plates are washed three times with distilled water to lyse the cells. EROD activity (pmol mg cellular protein per min) in each sample is measured... 

Reagents. The following buffers and solutions are needed for whole cell screening growth media Dulbecco s modified Eagle s medium (DMEM), 10% fetal calf serum (FCS), 100 units/ml penicillin, and 100 /ig/ml streptomycin, PBS 8 mM Na2HP04, 1.4 mM KH2P04, 137 mM NaCl, 2.7 mM KC1, pH 7.2. 

Cell Culture. KB cells were maintained in a humidified atmosphere of 5% carbon dioxide - 95% air at 37°C in the presence of modified Eagle s medium containing calf serum (10%), penicillin (100 jug/ml) and streptomycin (100 units/ml). Cells were routinely subcultured with 0.25% trypsin and stocks were discarded after twenty passages. All drugs were administered with fresh media 24 hours after subculture in the following concentrations TPA, 1.6 uM RA, 1.6 juM butyric acid, 2mM. Drug treatments were for 20-24 hours. Cells were harvested for enzyme assays with phosphate-buffered saline containing 0.05% EDTA and stored at -20°C in 0.32 M sucrose. 

Media frequently employed in the culture of continuous mammalian cell lines include Eagle s medium, MEM (Eagle, 1959) Eagle s medium modified by Dulbecco, DMEM (Dulbecco and Freeman, 1959) RPMI 1640 medium (Moore et ah, 1967) CMRL 1066 medium (Parker et al., 1957) and Ham s F12 medium (Ham, 1965). For the cultivation of adherent continuous cell lines the following basal media are suitable CMRL 1066, MCDB 411, DMEM. F12, MCDB 301, and IMDM. For non-transformed cells, the media DMEM, IMDM, MCDB 104, 105, 202, 401, and 501 are suitable (Freshney, 1992). Each of these basal formulations may be supplemented with serum or other specific proteins. 

The different forms of Eagle s medium are available commercially for example from Flow Laboratories or Gibco (Appendix 3). Complete 1 X MEM medium only requires the addition of glutamine, antibiotics and serum, and this is very suitable for small scale work in laboratories which lack facilities for preparing media. It is, however, a very expensive way of buying media if more than a litre or so is required per month. 

Growth media, i.e. media suitable for the continued culture of established cell lines, are prepared by dilution of the 10 X stock solutions. If the 10 X stock is made with salts present (i.e. if it is obtained commercially or if it is made from powder — see below) then it must be diluted with water. If, however, it is made as described in Appendix 1, Table 13 or 14, it must be diluted with BSS as indicated in Table 15. The latter table gives methods of preparing various minimal essential media supplemented with calf or foetal calf serum and in one case also with non-essential amino acids . The various media are all based on Eagle s MEM and have been developed to improve the growth of certain cell lines or strains as indicated. 

Cells are cultured in Eagle s MEM containing Earle s BSS or Hank s BSS and with 10% FBS. The life span could be increased with different media formulations, e.g. CMRL 1969> MEM> BME. 

Use the type of medium the cells have been growing in as control. Test 3-4 commercially available balanced media, such as F12/DMEM (Ham s nutrient mixture/Dulbecco s modified Eagle s medium), DMEM, IMDM (Iscove s modified Dulbecco s medium), HB-CHO (Hana Biological Chinese hamster ovary medium) or RPMI-1640 (Roswell Park Memorial Institute). 

Eagle s medium Any of a number of media used in tissue culture. 

RPMI 1640 media, Ham s F12K media, and Eagle s Minimal Essential Medium, MEME, respectively. All cell lines were grown in a tissue culture incubator supplied with 5% C02 and maintained at 37°C. 

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