S 570

The chemical elements that constitute the purified nanoparticles were determined with SEM-EDS. For that purpose, recovered pellets of selenium particles were analyzed with a scanning electron microscope (Hitachi S-570, Tokyo, Japan) with energy-dispersive X-ray spectra (SEM-EDS).

For SEM analysis, cells were planted on cover glasses (25 × 25 mm) which were placed in 6-well multiplies. After being pretreated with CA for 3 h, cells were stimulated with LPS for 12 h, cover slips removed, and cells fixed with 3% glutaraldehyde (room temperature) for 24 h. Fixed cells were rinsed with PBS and then dehydrated in EtOH (70%>80%>90%>95%>100%) and dried in a critical point dryer (Hitachi SCP-II). After coating with gold using an IB-5 ion coater (Eiko), cells were observed under SEM (S-570, HITACHI, Japan).
For TEM analysis, cells were fixed with 3% glutaraldehyde at 20°C for 48 h. Following fixing, cells were washed and dehydrated as before, and cells were embedded in Epon-Araldite mix solution and blocked at 60°C in a vacuum drying oven (Yamoto, DPF-31) for 36 h. First, semithin slides were made using an ultramicrotome (LKB-2088) and stained with 1% toluidine blue (1% borax) on a 60°C hot plate for 2 min. Then, ultrathin slices were made and stained with uranyl acetate and lead citrate. The cell microstructures were observed under TEM (JEM-1230, JEOL, Japan).

The freshly obtained blood sample (1 ml) was washed according to the procedure in Section 2.3.1. The purified hRBCs were diluted by 25 times with PBS. 0.2 ml of the diluted cell suspension was incubated with 0.8 ml OTC solutions of different concentrations for 3 hours under gentle shaking. Following incubation, the samples were centrifuged (2000 rpm×5 min) and the supernatant was discarded. Fixation was performed by addition of 2.5% glutaraldehyde and 12 h incubation. The fixed samples were washed with 0.1 M phosphate buffer for more than 3 h. Then, the sample was fixed in osmium tetroxide (1%) for 1∼1.5 h and washed in double-distilled water for 2 h. Dehydration was done with increasing concentrations of ethanol (50%, 70%, 80%, 90% and 100%) twice. The ethanol was displaced by isoamyl acetate (100%) for 15 min (twice). Finally, the sample was dried with the conventional critical point drying method, platinum-coated with ion sputtering coater (Eiko, IB-5) and then observed with a scanning electron microscope (Hitachi, S-570) to investigate the effect of OTC on the morphology of hRBCs.