Fluorescent Uptake of Hst1 by Osteogenic Cells

To analyze the uptake and localization of fluorescently labeled Hst1 (F-Hst1) by osteogenic cells, cells from a semi-confluent cell culture were transferred into a 48-wells plate at a density of about 3.5 × 10⁴ cells/well and cultivated at 37°C for at least 24 h. Subsequently, the cells were washed once with DPBS (Dulbecco’s PBS, Gibco) after which serum-free medium was added. Two micrometer F-Hst1 was added and after a 1 h period, the cells were washed four times with DPBS containing 0.9 mM Ca²⁺ and 0.5 mM Mg²⁺. As a negative control, incubations without F-Hst1 were included. The cells were studied by the EVOS-FL microscope with a 20x objective with a phase contrast setting and a “Cy-5 light cube” with a 628/40 excitation filter and a 692/40 nm emission filter. Digital photographs were recorded by a computer integrated in the microscope.
To more precisely observe the uptake and localization of F-Hst1, the cells were further studied using a LEICA TCS SP8 confocal laser scanning microscopy (CLSM) system as previously described (Ma et al., 2020 (link)). Before incubation with F-Hst1, cell nuclei and membrane were stained with NucBlue^TM live cell stain (Life Technologies, Grand Island, NY) and PKH67GL (Sigma–Aldrich, MO, United States) respectively, following manufacturer’s instructions.