To indicate [Ca2+]i signaling, cell networks were incubated in a humidified incubator for 45 minutes with 10 μM Fura-2 AM medium (Molecular Probes, Eugene, OR) and then rinsed with fresh working medium (α-MEM without phenol-red supplemented with 2% FBS and 2% CS) three times. The slide was mounted into a custom-built parallel plate flow chamber for laminar fluid flow stimulation (Fig. 1C ). The flow chamber was mounted on an inverted fluorescence microscope (Olympus IX71, Melville, NY) and left undisturbed for 15 minutes, which has been shown to be sufficient for bone cells to recover from disturbance and to generate repetitive [Ca2+]i responses (45 (link)). A magnetic gear pump (SiLog, Micropump, Inc., WA) was connected to the chamber to run the fresh working medium through the chamber with a desired steady flow rate.
The [Ca2+]i responses of bone cell networks under fluid flow stimulation were recorded with a high-speed CCD camera (ORCA-ER-1394, Hamamatsu Photonics K.K., Hamamatsu City, Japan) for a period of total 10-minutes, one minute for baseline and 9 minutes after the onset of fluid flow. Fura-2 340 nm/380 nm ratio images were used to obtain the dynamic history of [Ca2+]i by measuring the average image intensity of each cell using MetaMorph Imaging Software 7.0 (Molecular Devices, Downingtown, PA). The intensity of [Ca2+]i for each cell was normalized by its corresponding baseline.
The [Ca2+]i responses of bone cell networks under fluid flow stimulation were recorded with a high-speed CCD camera (ORCA-ER-1394, Hamamatsu Photonics K.K., Hamamatsu City, Japan) for a period of total 10-minutes, one minute for baseline and 9 minutes after the onset of fluid flow. Fura-2 340 nm/380 nm ratio images were used to obtain the dynamic history of [Ca2+]i by measuring the average image intensity of each cell using MetaMorph Imaging Software 7.0 (Molecular Devices, Downingtown, PA). The intensity of [Ca2+]i for each cell was normalized by its corresponding baseline.
