Apoptosis analysis in Sf-9 insect cells

Description

Infection by many viruses triggers apoptosis. This can benefit either the virus or the host, and accordingly many hosts and virus have evolved mechanisms and counter mechanisms designed to promote and inhibit apoptosis. Thanks to the methods described here, several apoptosis-related genes have been identified in both shrimp and WSSV, and we expect that more related genes will be identified in the future. This section introduces three methods for testing the anti- or pro-apoptosis activities of genes in Sf-9 cells.

Brief protocol

Cell culture

Spodoptera frugiperda (Lepidoptera: Noctuidae) Sf-9 insect cells are maintained at 27°C in Sf-900 II SFM medium (Invitrogen) supplemented with or without 10% heat-inactivated fetal bovine serum (Hyclone Laboratories).

Gene cloning

Your favorite genes could be cloned into the two vectors by PCR or other standard cloning procedures.

DNA Transfection and protein expression

Sf-9 cells are seeded in six-well plates (1 x 10⁶ cells/well) and wait for 30 min until the cells adhere to the plates. Then the cells are reacted with transfection mixtures containing 4 μg of the plasmid DNA and 4 μ of Cellfectin reagent (Invitrogen) for 30 min. The transfection mixtures are discarded, replacing with fresh SFM medium, and the transfected cells are returned to 27°C incubator. At 18-20 h post-transfection, the cells are heat shocked in a 42℃ water bath for 30 min to induce the expression of the transfected genes, and then returned to 27℃. At 4 – 6 h after heat shock, the transfected cells are washed with PBS, and the expressed protein can then be assay by Western blot detecting with anti-V5 or anti-FLAG antibodies. Or protein activity assay can be performed.

References

1. Clem RJ, Miller LK. Control of programmed cell death by the baculovirus genes p35 and iap. (1994) Mol Cell Biol 14: 5212-5222.

Some of our publications that have used this platform

1. Leu JH, Kuo YC, Kou GH, Lo CF. (2008) Molecular cloning and characterization of an inhibitor of apoptosis protein (IAP) from the tiger shrimp, Penaeus monodon. Dev. Comp. Immunol 32:121-133.
2. Leu JH, Wang HC, Kou GH, Lo CF. (2008) Penaeus monodon caspase is targeted by a white spot syndrome virus anti-apoptosis protein. Dev Comp Immunol 32: 476-486.