Engraftment analysis by direct cell counting. (A) Example of FACS separation and counting of eGFP⁺ and eGFP⁻ cells extracted from bone marrow and spleen of untreated controls (left) and treated mice (right). The sorting gates were set to preferentially select mononuclear cells. The percentage of donor cells was calculated selecting the fluorescent population based on the fluorescence intensity (FL1-H) versus forward scatter (FSC-H). (B) Confocal microscopic images of 10-μm bone sections from untreated (Brtl) and treated (Brtl + IUT) mice. Blue DAPI counterstaining reveals nuclei of all donor and host cells. Green eGFP⁺ donor cells are visible in the treated sample in the region surrounding the trabeculae at expected locations of osteoblasts and in the cortical region occupied by osteocytes (eGFP⁺ osteocytes are indicated by red arrows). The imaging was performed in a TCS SP2-Leica confocal microscope with 40×/1.25 NA oil objective; 488 nm excitation and a 500-540 nm emission filter were used for eGFP, and 364 nm excitation and 400-480 nm filter were used for DAPI. (C) Phase contrast (left) and GFP fluorescence (right) images of primary osteoblasts growing from minced femur and tibia chips, obtained with the DMIL-Leica microscope (10×/0.22NA objective).