Phosphate → Leukotrien C4

Participants

Reaction Steps

5.0 μg of plasmid DNA (pMON 23962) containing the LTC4 synthase gene was mixed with 200 ng of linearized baculovirus DNA (Pharmingen, San Diego, Calif.; Catalog # ) in an eppendorf tube, using the calcium phosphate transfection method described in Summers, M. D., and Smith, G. E. (1987), A manual of methods for baculovirus vectors and insect cell culture procedures, Texas Agriculture Experiment, Station Bulletin no. 1555. To the the same tube, 0.75 ml of transfection 15 buffer (25 mM HEPES, pH 7.1, 140 mM NaCl, 125 mM CaCl2) was added and the tube was vortexed. Sf9 insect cells grown in ITF media [IPL41 media (GIBCO-BRL) plus 10% fetal bovine serum (JRH Biosciences) plus 1X tryptose phosphate broth (GIBCO-BRL)] in a spinner flask were seeded in a 25 cm2T-flask at 2.0×106 cells/flask. The cells were allowed to attach for 1 hour at RT (22-27° C.). At the end of 1 hour, the media from the flask was removed and replaced with 0.75 ml of Grace's medium (GIBCO-BRL) containing 10% fetal bovine serum (JRH Biosciences) and 1x antibiotics [Antibiotic-Antimycotic, Cat.# 15240-039 (GIBCO-BRL)]. DNA in the transfection buffer was added dropwise to the the Grace's media (GIBCO-BRL) in the cell culture flask. The flask was incubated at 27° C. for 4 hours in an incubator such that the cells were covered with the liquid in the flask. At the end of 4 hours, the medium from the flask was removed by a pasteur pipette. To the flask was added 5 mls of the ITF media containing antibiotics and the cells were gently washed by rocking the flask. The media was removed and the process repeated two more times. The cells were then incubated at 27° C. in 5 mls of the ITF media containing the antibiotics in the T-flask. After seven days of incubation, the cells were resuspended in the media contained in the flask and transferred to a 15 ml screwcapped tube. The tube containing the cells was spun at 3000 rpm for 10 minutes in a Beckman GPR tabletop centrifuge with the brakes off. The cell-free supernatent was transferred to a new tube and the cell pellet assayed for LTC4 synthase activity.

Provenance & Evidence