Dmi4000b inverted microscope

Osteoblast migration assays were performed using Culture-Insert μ-Dishes as described by the manufacturer (Ibidi). Primary WT or Pyk2-KO osteoblasts were seeded into the inner well of the μ-Dish and incubated at 37°C and 5% CO₂ overnight. Alternatively, prior to plating in migration chambers, primary osteoblasts were infected with adenovirus expression constructs for Pyk2 using multiplicity of infection (MOI) of 300 per virus for 3 days. The migration chamber inserts were removed, unattached cells were rinsed off, and osteoblasts were incubated with alpha-MEM containing 10% serum for 6 hours. The migration of cells into the clear zone outside of the plating area was quantified microscopically. For migration assays involving MKs, 1x10⁵ osteoblasts were first plated into migration chambers and incubated overnight. The following day, chamber inserts were removed and osteoblasts were imaged before adding 1x10⁴ MKs to each well. Osteoblasts plus MK co-cultures were then incubated for an additional 6 hr, after which the cells were fixed in 4% formaldehyde. Osteoblast migration distance was determined by microscopic imaging using a Leica DMI4000B inverted microscope with attached digital camera. Final analyses were performed using Image Pro software (Media Cybernetics, Bethesda, MD).