ABSTRACT
The spruce budworm (C.fumiferana) cell line is usually called as IBRI-Cf1 cell line which is reared in Grace’s insect tissue culture medium (pH 6.2) supplemented with 0.25 per cent tryptose broth, 10 per cent fetal bovine serum, and 0.002 per cent penicillin-streptomycin in 75 cm2 polystyrene flasks. Cultures are initially seeded at cells/ml and subcultured every 5-7 days (Gole et al. 1987). For an assay whole cells are washed 2 times with a buffered solution of physiological saline and resuspended in physiological saline containing 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) and the test compound. Washed membrane preparations are obtained by homogenizing washed whole cells in 110 mM Tris/1 mM dithiothreitol (DTT)/ 1 mM EDTA (pH 7.0) with 30 strokes in a glass-teflon homogenizer. Following centrifugation at 900 g for 5 minutes the supernatant fraction is centrifuged at 28,000 g for 10 minutes and the resulting pellet washed with 10 mM TRIS/1 mM DTT/ 0.4 mM EDTA (pH 7.0). The final pellet is resuspended in above solution and incubated at 35 °C for 20 minutes in 200 µl reaction mixture containing 75 mM Trisacetate (pH 7.2), 0. 05 mM GTP, 0.1 mM IBMX, 30 mM magnesium acetate, 1.5 mM ATP and the tissue preparation. The reaction is terminated by adding 1 ml 0.4N perchloric acid. Samples are then assayed for cyclic AMP (Gole et al. 1987). This procedure has been used for a natural diterpene, forskolin, to show insensitivity against adenylate cyclase. Obviously such an assay can be used for other phytochemicals as well to demonstrate the sensitivity towards insect physiological systems.
