Effects of CaR stimulation on primitive hematopoietic cell activity in vitro. (A) LK cells were loaded with indo-1 dye (2 μg/mL) as a fluorescent probe to mark intracellular Ca²⁺ concentrations. CaCl₂ (1.5mM) was added as an exogenous source of Ca²⁺ ions to induce a response. The response in LKS⁺F⁻, LKS⁺F⁺, and LKS⁻F⁺ subpopulations was then analyzed with FlowJo software. Arrow indicates the addition of CaCl₂ stimulus. Green indicates control; red, Cinacalcet (n = 4 from 4 independent experiments). (B) BM MNCs treated with Cinacalcet or ethanol control were assessed for in vitro growth potential with the use of the culture colony-forming unit assay (n = 10 from 5 independent experiments). (C) LKS⁺F⁻, LKS⁺F⁺, and LKS⁻F⁺ cells were seeded on a confluent stromal layer of supportive OP9 cells in serial dilutions and cultured at 33°C and 5% CO₂. Cobblestone areas were scored on week 5 (*P < .05; n = 9 for LKS⁺F⁻; n = 7 for LKS⁺F⁺; n = 3 for LKS⁻F⁺ from 5 independent experiments; error bars represent SEMs). CFC indicates colony-forming cell.