The cells (usually 1.5–2 × 106/ml) are infected with amplified, recombinant baculoviruses the next day. ���� K�N*��iDzR;��Iǥ�N_�T�V��`�������X�-�`4p� � Quickprep miniprep using an isolation filter may work, but DNA must be sterile. Allow the cells to attach by incubating at 28°C for 20 min. The standard sorting conditions used for mammalian cells proved to be unsuitable, resulting in post-sorting viabilities below 10% for both cell lines. Protocol for: Madhi SA, Polack FP, Piedra PA, et al. Cell densities in excess of 8.3 x 10^6 cells/ml can be achieved with suspension cultures of Sf9 cells using Insect-XPRESS™ Medium and an excess of oxygen. Zhang PF, et al. The Sf9 cells are highly susceptible to baculovirus, which is widely used Protocol for MIR 6100, 6104, 6105, 6106 Quick Reference Protocol, ... developed for high-performance transfection of plasmid DNA into insect cells such as: Sf9, High Five™ (BTI-TN-5B1-4), and Drosophila melanogaster Schneider’s 2 (S2 or D. Mel. The latter is resuspended in lysis buffer and spun down again (200,000g) to 45 min at 4°. By continuing you agree to the use of cookies. Integral membrane proteins have a critical role in fundamental biological processes; they are major drug targets and therefore of high research interest. Glass culture flasks with steel closures of various sizes (10 ml to 2 liters) (Bellco) are used for suspension cultures. See page 2 to view a procedure for transfecting Sf9 or Sf21 with Baculovirus DNA. This kit is a second-generation method that replaced our earlier Sf9 Insect Cell HCP ELISA Kit (F020). We obtain serum from Atlanta Biologicals (Atlanta, GA). Biologicals 22: 205-213, 1994. Sf9 (Spodoptera frugiperda ovary) insect cells (Pharmingen, San Diego, CA) are grown in suspension in a shaking incubator (Innova 4000, New Brunswick Scientific, Edison, NJ) in IPL-41 medium (Gibco-Life Technologies, Rockville, MD) supplemented with 10% fetal bovine serum, 0.1% Pluronic F68 (Gibco-Life Technologies), gentamicin (50 μg/ml) and Fungizone (50 μg/ml) (Fungizone and gentamicin were from Gibco or the Tissue Culture Center, Washington University). Dounce ~50 strokes on ice using the B pestal. 4 0 obj Remove and discard culture medium. ESF-921 Delta Series methionine-deficient insect cell culture medium (Expression Systems, 96-200). Sf9 Insect Cells. For Sf9 cells: Plate cells at a density of 0.5 x 106 cells/ml. The culture is split every 2–3 days to maintain a density of 10–2.5 × 106 cell/ml as measured with a hemocytometer. General protocols for inducing apoptosis in cells Apoptosis may be induced in experimental systems through a variety of methods. �m �#$��! stream Here, we provide simplified protocols that facilitate the generation of high-quality virus and initial expression analysis for integral membrane protein targets utilizing the baculovirus-mediated expression system in insect cells.

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