Production of Conditioned Medium containing Wingless protein from S2 cells

- Grow heatshock wingless S2 cells at 25 degrees centigrade in a tissue culture flask to approximately 2.5 x 106 cells/ml.
- Heat shock the cells at 37 degrees C for 30 minutes in the same flask
- Move the cells back to 25 degrees for 40 minutes to allow the cells to recover
- Remove the cells by pipeting, pellet them by spinning at 1200rpm and wash them 3 times with serum free medium to remove serum components.
- Change medium to serum free (if desired, reduce the original volume by 10 fold to enrich for wg)
- Collect conditioned medium 3-5 hours
- Spin at 1200 rpm for 10 minutes to pellet the cells
- Remove the medium and spin at 100,000 g's for 30 minutes at 4 degrees C to pellet any insoluble material
- Remove the supernatant which contains soluble wingless protein. We concentrate the medium using Amicon Centricon 30 or Centriprep30 concentrators.
- Either use immediately or flash freeze in liquid nitrogen and store and -70 degrees C