END2 and HepG2 cells were incubated (37ºC, 96 hours) in T-flasks containing DMEM media and FBS (10%v/v). The resulting condition media was decanted, filter-sterilized through a 0.22 µm filter, and stored at -80ºC until further use. The thawed conditioned media was used to culture mouse ES cells in six-well plates that were incubated further (37ºC, 96 hours).
As a control, six-well plates of mouse ES cells were incubated (37ºC, 96 hours) in standard basal media. The cells were examined using a DM1L microscope to determine if the conditioned media had any effect on morphology. The media was removed from all plates after four days of culture in the conditioned media, an osteogenic media was added, and the cells were incubated (37ºC, 20 days). The cells were stained with Alizarin Red S and examined using a microscope to establish if the conditioned media had any effect on bone-nodule formation.
To determine if there were any proteins in the conditioned media that could be having an effect on cell differentiation, samples of both END2- and HepG2-conditioned media were concentrated 1,000-fold using Millipore’s Centricon® plus-70 centrifugal filter (5 kDa MWCO). These protein samples (150 µg) were run under standard electrophoresis conditions as preparative 2-D DIGE gels with the samples run on the first-dimension gel (nonlinear pH 3–10 IPG strip). The IPG strip was then applied to the second-dimension 18 cm x 16 cm pre-cast Bis-Tris 4-12% polyacrylamide gel and the gels were then stained with colloid Coomassie Blue.
A second set of 2-D DIGE gels was then run where the concentrated END2- and HepG2-conditioned media samples (50 µg) were mixed with Cye dyes. The END2- conditioned media was labeled with Cy5, the HepG2-conditioned media was labeled with Cy3 (and vice versa) in a reciprocal manner and both conditioned-media samples were mixed and labeled with Cy2.
The gels were then transferred to Dyversity, a fully automated CCD-based 2-D gel image analysis system (Figure 1) rather than a laser scanner for imaging.
The gels were imaged using the Cy dye lighting module and 8x8 binning for an exposure time that was optimized for maximum dynamic range. The image was analyzed with image-analysis software and common spots were excised and trypsin digested. Proteins were identified using MALDI-TOF for peptide mass analysis, and the information was analyzed using MASCOT for database searches to identify proteins.