After 3 days of culture in NGF supplemented medium cells were viable and extended neurites.
After removal of the medium, cells were lysed and cAMP accumulation was determined using the SMP004 kit from PerkinElmer according to the instructions of the manufacturers.
For RNA isolation from culture medium, cells were seeded as described above.
When cell outgrowth occurred (appearance of cell clumps and acidification of medium), cells were expanded twofold per stimulation.
After washing with pre-warmed cell culture medium, cells were left to rest for 30 min at 37°C and 5% CO2, in cell culture medium.
After release of cells from α-factor block with YPAD medium, cells were arrested in metaphase due to repression of Gal1-CDC20 expression [59].
After 18 hr of incubation in complete medium, cells were re-plated and stimulated in the presence or absence of 100 nM C3a for 6 hr.
At the end of the culture period (72 hours for cells in proliferation and 36 hours for cells in differentiation medium), cells were rinsed, trypsined and numbered.
Following removal of [3H]NLX containing binding medium, cells were washed twice with PBS (37°C), collected and [3H]NLX in cells were counted by scintillation spectrometry.
After removal of the culture medium, cells were treated with 1x Trypsin/EDTA (Gibco) for several seconds, leading to selective detachment of myotubes.
After 30 minutes of incubation at 37°C, 5% CO2 in 500 µl of growth medium, cells were seeded at 1×105 cells/ml.
