4.17. Osteogenic Differentiation

Marrow cells were cultured in a T75 flask for 7 days and then trypsinized, counted, passaged, and plated in osteogenic media (Alpha-MEM, 0.5% β-glycerophosphate, 0.1% ascorbic acid-2-phosphate, 1% L-glutamine, 1% antibiotic-antimycotic) at density of 200,000 cells per well in a 6-well plate. At day 14, cells were stained with ALP as described or Alizarin Red S (ARS) to image mineralization. For ARS staining, cells were washed gently with PBS and fixed with 4% PFA for 60 minutes under slow rotation. Filtered 1% Alizarin Red S (Millipore, A5533) was added to the cell monolayer for 15 minutes in the dark at 25°C. Plates were washed 3–4 times with distilled water until background staining was removed. ALP and ARS plates were imaged using an inverted conventional brightfield microscope (BioTek, Lionheart FX) affixed with RGB imaging cubes. Stitched imaged were acquired using a Phase Plan Fluorite 1.25x air objective, registered using the blue channel, and stitched with 10% overlap. Stitched images were analyzed for staining quantity using Fiji ImageJ2. ⁴⁹ Only the blue channel was used for analysis. A 560 × 560-pixel circular ROI was created to capture the whole plate but exclude edge artifacts. Image thresholds were set to 21/124 and 139/158 for ALP and ARS, respectively. Images were then measured for percent of ROI with positive staining.