Our study compared the LDN-193189 oscillatory pump method to the orbital platform method in both the Pyk2 primary calvarial osteoblasts and the FAK calvarial osteoblast clones. We determined that there was not a significant difference between the methods as measured by the response of osteoblasts to up-regulate OPN expression and to increase c-Fos and COX-2 protein levels. We did observe a difference between the two methods when analyzing the phosphorylation of ERK over time. Using the oscillatory pump methods, we determined that in both primary calvarial osteoblasts and in osteoblast clones the peak of ERK phosphorylation occurred at 5 minutes of FF. Using the orbital pump method we observed this peak in ERK phosphorylation to occur at 15 minutes. Because the oscillatory pump methods requires more manipulation of the glass slide in media while being placed in the parallel plate flow chamber, we suggest that these osteoblasts are being stimulated for approximately 10 minutes prior to turning on the pump. In contrast, the osteoblasts on the 6 well plates are not manipulated prior to starting the orbital platform shaker. Therefore, the extra manipulation of the slides may be a possible reason why we observe a difference in timing of ERK phosphorylation between the two methods. Interestingly, we found that the fold difference at apex of ERK phosphorylation in not significantly different between the two methods. Secondly, the oscillatory pump experiments were conducted at 0.5 Hz while the orbital platform experiments were performed at 2 Hz, which may also contribute to the difference we observe in ERK phosphorylation between the two methods. Finally, the oscillatory pump method generates oscillatory fluid flow while the orbital platform method generates dynamic fluid flow. The differing types of FF could also be a possible explanation for the differences in ERK phosphorylation we observed. In our study we did observe a difference in OPN expression in immortalized calvarial osteoblasts when placed on glass slides as compared to those plated onto 6 well plates. There was a significant 4–10 fold increase in OPN expression when osteoblasts were plated on 6 well plates as compared to cells plated on glass slides. However, we did not observe this increase in OPN expression in the primary calvarial osteoblasts. Therefore, we speculate that the increased OPN expression observed in the immortalized osteoblasts may be due to a clonal effect, though this hypothesis has not yet been tested. Other labs have also used an orbital platform like method to mechanically stimulate cells. Kido et al. mechanically stimulated osteoblasts using a horizontal shaking apparatus fixed inside a tissue culture incubator to study how mechanical stimulation affects interleukin-11 expression. They conclude that this method generates FSS that is similar to a low-magnitude, oscillatory FSS generated by the parallel-plate system.