In VitroCell.Dev.Biol.--Animal 34:629-630,September 1998 © 1998Society for In VitroBiology 1071-2690/98 $05.00+0.00 Letter to the Editor CULTURE OF MOSQUITO CELLS IN EAGLE'S MEDIUM Dear Editor: Aedes albopictus mosquito cells derived from the ATC-15 line of Singh (1967) have been extensively used in production of somatic cell mutants (Gerenday et al., 1997), expression of transfected genes (Fallon, 1989; Shotkoski and Fallon, 1994), and most recently, iso- lation and characterization of factors involved in the inducible im- mune response (Hernandez et al., 1994; Yoshiga et al., 1997). Al- though Ae. albopictus cells are often maintained in "insect" tissue culture media, such as the formulation of Mitsuhashi and Maramo- rosch (1964) which contains lactalbumin hydrolysate and yeastolate, the ability of mosquito cells to thrive in vertebrate tissue culture media under a standard 5% CO2 atmosphere was noted in the original description of ATC-15 cells (Singh, 1967) and has facilitated com- parative studies between mammalian and mosquito cells, Apart from the serum component, vertebrate tissue cuhure media such as Eagle's medium are completely defined, a feature that fa- cilitates development of selective conditions for recovery of somatic cell mutants and use of radioisotopes as metabolic precursors in biochemical studies. In particular, the C7-10 Ae. albopictus cell line used in current analyses was derived from cells that had been grad- ually adapted from the Mitsuhashi and Maramoroseh (1964) medium to Eagle's (1959) vertebrate tissue culture medium supplemented with L-glutamine, nonessential amino acids, antibiotics, and 5% fetal bovine serum (Sarvcr and Stollar, 1977; reviewed in Fallon and Stol- lar, 1987). Preparation of this medium by combining Earle's salts, essential and nonessential amino acids, vitamins, glutamine, anti- biotics, sodium bicarbonate, and fetal bovine serum has been de- tailed previously (Fallon, 1989). In this letter, we describe a simpli- fied preparation protocol based on the commercially available powdered or liquid formulations shown in Table 1. In a first application, we used a powdered Eagle's minimal essen- tial medium (MEM) from Sigma Chemical Co. (St. Louis, MO) con- taining Earle's salts, L-glutamine, and nonessential amino acids (Ta- ble 1) as a base. Briefly, 10 g of the powdered Sigma medium was dissolved in 875 ml of distilled water with constant stirring. To arrive at equivalent concentrations of key components, relative to the earlier formulation (Fallon, 1989), additional nonessential amino acids and glutamine, vitamins, D-glucose, antibiotics, and sodium bicarbonate (Table 2) were added, as detailed in Table 3. After adjusting the pH to 6.8 with I N HCI or 1 N NaOH, the volume was adjusted to 1000 mL, and the medium was sterilized by filtration and stored as 95-ml aliquots at 4 ° C. Prior to use, heat-inactivated fetal bovine serum was added to a final concentration of 5%. When C7-10 cells were maintained in the Sigma-based medium (Table 3), cell growth mea- sured with a Coulter electronic cell counter was indistinguishable from that in medium prepared from components as described pre- viously (Fallon, 1989). TABLE 2 SOURCES AND CATALOG NUMBERS OF SUPPLEMENTS USED IN PREPARING CULTURE MEDIA Component Supplier Distilled water Sodium bicarbonate MEM nonessential amino acids MEM vitamin solution L-glutamine Penicillin/Streptomycin D(+)glucose (dextrose) m (7.5% wt/vol; GIBCO BRL #25080) (10 mM = 100X, GIBCO BRL #11140) (100X; GIBCO BRL #11120) (200 mM = 100X; GIBCO BRL #25030) (10,000 U/ml; 10,000 ~g/ml; GIBCO BRL #15140) (10% wt/vol; GIBCO BRL #15023; Sigma G-7021) TABLE 1 COMMERCIAL FORMULATIONS AND CATALOG DESCRIPTIONS OF CULTURE MEDIA Formulation Description Powder Powder Liquid Liquid Sigma M-0643: Eagle's Minimum Essential Medium (MEM); with Earle's salts, L-glutamine, and nonessential amino acids; without sodium bicarbonate GIBCO BRL #61100 (MEM with Earle's salts and L- glutamine; without sodium bicarbonate GIBCO BRL #11095 (1 × MEM with Earle's salts and L-glutamine) GIBCO BRL #51200 (1 × MEM with Earle's salts; without phenol red, without L-glutamine) TABLE 3 ADDITIONS TO MEDIA TO MAKE THEM COMPARABLE TO THOSE USED IN FALLON (1989) Source,catalog number, and amount Characteristic Sigma GIBCO GIBCO GIBCO or ingredient M-0643 61100 11095 51200 Weight or volume (prepackaged) 10 g 9.5 g 1 L 1 L Distilled water 875 ml 875 ml -- -- Glucose 10 ml 10 ml 10 ml 10 ml Glutamine 10 ml 10 ml 10 ml 20 ml Vitamins 10 ml 10 ml 10 ml 10 ml Nonessential amino acids 10 ml 20 ml 20 ml 20 ml Antibiotics 10 ml 10 ml 10 mt 10 ml Sodium bicarbonate 29.3 ml 29.3 ml -- -- 629