Nucblue fixed cell readyprobe reagent

MC3T3-E1 cells were plated onto glass coverslips at a density of 5 × 10⁵ cells/well in 24 well plates along with 3 mg of nanopowders. Cells were cultured for 7 days in differentiation media along with nanopowders. At the end of 7 days, cells were washed 1× with PBS to remove excess particles and fixed for 5 min with 4% paraformaldehyde. Cells were then washed 3× in PBS for 5 min each. Fixed cells were incubated for 1 h in 1% bovine serum albumin with 0.1% Triton-X. After blocking, cells were stained with Alexa Fluor 568 Phalloidin (1:400) (Molecular Probes) for 1 h at room temperature. After incubation, coverslips were rinsed in 1× PBS and washed for 3 × 5 min in 1× PBS. Cell nuclei were then counterstained using NucBlue fixed cell ReadyProbe reagent (Molecular Probes) for 20 min. Images were obtained using a Zeiss LSM 710 confocal microscope (UIC core imaging facility).

RAW264.7 cells seeded in coverslips laid at the bottoms of the 24-well plates were differentiated for 7 days in media containing 1 mg/well of Se-HAp nanoparticles. Cells on coverslips were then first washed with 1x phosphate buffered saline (PBS) to remove the nanoparticles that had not been endocytosed, then fixed for 5 minutes in 4 wt.% paraformaldehyde, then washed with 1x PBS and incubated in the dark for 30 minutes with Alexa Fluor 568 Phalloidin (1:400) (Molecular Probes) to stain f-actin filaments and OsteoImage™ (Lonza) to stain Se-HAp particles. After the incubation, the coverslips were rinsed in 1x PBS and washed in the dark for 3 × 5 minutes in 1x PBS. Cell nuclei were then stained using NucBlue fixed cell ReadyProbe reagent (Molecular Probes) for 20 minutes. Images were acquired on a Zeiss LSM 710 confocal microscope.

RAW264.7 cells seeded at 5.5 x 10⁵ cells/well in 24-well plates and differentiated for 7 days in media containing 5 mg/well of CP nanoparticles. Cells on coverslips were then first washed with 1x PBS to remove the nanoparticles that had not been endocytosed, then fixed for 5 minutes in 4 wt% paraformaldehyde, then washed with 1x PBS and incubated with Alexa Fluor 568 Phalloidin (1:400) (Molecular Probes) and OsteoImage^™ bone mineralization staining agent (Lonza) for 30 minutes (for HAP, DCP and ACP) and 1 hour (for CPP), according to the protocol described by Lonza. After incubation, coverslips were rinsed in 1x PBS and washed for 3 x 5 minutes in 1x PBS. Cell nuclei were then counterstained using NucBlue fixed cell ReadyProbe reagent (Molecular Probes) for 20 minutes. Images were acquired on a Zeiss LSM 710 confocal microscope (UIC core imaging facility).