These observations suggest that fluorescence intensity depends, at least in part, on cell type, that is, possibly related to nuclear size as well as other factors [10]. We also examined whether 50μM verapamil, which blocks ABC transporters, decreased the fluorescence intensity. However, verapamil had only a minimal effect on the fluorescence intensity of IEC-6 cells (Figure 4(a)). The flow cytometric analysis also Selleck GSK2656157 demonstrated that fluorescence intensity was dose dependent

of Hoechst 33342. Interestingly two peaks were observed in IEC6 cells incubated with 100ng/mL Hoechst 33342, suggesting that fluorescent intensity may not be uniform even in the same type of cells, probably due to the heterogeneity of the IEC-6 cells in Inhibitors,research,lifescience,medical the cell cycle. Figure 4 (a) Dose response relationship between Hoechst 33342 and fluorescence intensity in the presence or absence of 50μM verapamil in IEC-6

cells, (b) Hoechst Inhibitors,research,lifescience,medical 33342 dose response for fluorescence intensity in IU-937 cells, and (c) FACS analysis … We also investigated whether the way in which Inhibitors,research,lifescience,medical frozen tissue sections were prepared might have an effect on the fluorescent intensity of the cells. To simulate the preparation of frozen tissue sections we fixed, dehydrated and froze Hoechst 33342-stained IEC-6 cells, and then compared the fluorescence intensity before and after treatment. However, this treatment resulted in only a slight increase, rather than decrease, in fluorescence intensity (Figure 5). Figure 5 Effect of fixation, dehydration, and freezing of Hoechst 33342-stained IEC-6 cells on fluorescence intensity. IEC-6 cells stained with 100ng/mL Hoechst 33342 were observed by both phase contrast and fluorescent microscopy before ((a), (b)) and … In the next step we prepared Dio-labeled and Hoechst

33342-incorporated PLGA particles. Inhibitors,research,lifescience,medical The mean particle Inhibitors,research,lifescience,medical size and zeta potential were 333.8nm and −2.14mV, respectively (Figures 6(a) and 6(b)). The concentration of Hoechst 33342 in the supernatant of PLGA emulsion was 2.8μg/mL, suggesting that 14μg of Hoechst 33342 was contained in the aqueous phase. Because we used 20μg of Hoechst 33342 in total, the % entrapment of Hoechst 33342 was calculated as 30%. We observed the time-dependent increase of Hoechst 33342 concentration in the in vitro release experiment (Figures 6(c) and 6(d)). Figure 6 Distribution in the Adenylyl cyclase diameter of Dio-labeled and Hoechst 33342-incorporated PLGA particles. (a) The particles were pictured under fluorescent microscopy. (b) The size bar represents 5μm. (c) Standard curve for measuring Hoechst 33342 concentration. … Particles were administered to the mice by one of three different methods: (i) direct injection into the femoral muscle, (ii) intravenous administration, or (iii) intraperitoneal injection. Frozen tissue sections from the femoral muscle revealed nuclear staining with blue fluorescence around the green particles and lack of nuclear staining in the muscle away from the particles (Figures 7(a) and 7(b)).