To study the stalked cell response to carbon starvation, a population of swarmer cells was allowed to proceed through the cell cycle for 60 min and transition into stalked cells in complete medium, before removing the carbon source and incubating for 15 min in M2 medium.
Then add the flour, water and stock cubes and stir in over a medium heat, allowing to cook for a further 5 minutes before removing the thyme.
The cultivated cells were harvested by centrifugation and washed with distilled water to remove the medium components before being lyophilized.
Cells were then pelleted from 1 ml cultures by centrifugation to remove the growth medium before transferring to fresh LB medium in the presence or absence of 3.0 M NaCl, 3.0 M sorbitol and 1% H2O2.
Then G418 was added back to the culture medium and the cells were passaged for an additional 4 weeks before removing G418 from the culture medium.
The three types of macrophages were seeded at 1×10 cells/ml, cultivated overnight before removing non-adherent cells, replacing the medium, and cultivating with or without LPS (100 ng/ml).
Nine days before implantation, doxycycline was removed from the medium to allow BMP-2 expression, which was confirmed using an ELISA kit (R&D Systems, Minneapolis, MN).
It is clear that Yap8 levels were enhanced during this period (Fig. 1B, left panel) in contrast to what does occur if As was completely removed from the medium before CHX treatment (Fig. 1B, right panel).
At least 72 h before each experiment STX140 was removed from the medium, and the cells were washed and cultured in fresh untreated medium.
