Effect of Carbon Nanotubes on Erythrocytes Figure 4. Effect of treatment with control and acid functionalized SWCNTs on hydrophobicity of erythrocyte membrane. Erythrocytes (56106 /ml in PBS+1%FBS at 37uC) from Swiss (panel A) and C57Bl6 (panel B) mice were incubated with 25 mg/ml of SWCNTs and AF- SWCNTs for 1 h. Cells were washed and incubated in PBS with 4 mM ANS for 20 min in dark at 37uC, after which fluorescence spectra was determined on a fluorimeter. doi:10.1371/journal.pone.0022032.g004 was generalize or was selective for erythrocytes of specific age groups. To determine the susceptibility of erythrocytes of different age groups, we used a technique that we recently developed to enumerate circulating erythrocyte cohorts of different age groups [9]. In this technique that involves a two step in vivo biotinylation of circulating erythrocytes, erythrocytes of different age groups can be identified as a biotin negative population (young erythrocytes, age ,10 days), a biotin high population (Old erythrocytes, age.40 days) and a biotin low population (erythrocytes of age between 10 to 40 days). Results in Figure 8A show that just 3 h after a single dose of AF- SWCNTs, the proportion of old erythrocyte (age.40 days) in blood fell from 7.3% to 5.8%, whereas the proportion of young erythrocytes (age,10days) increased from 36.19% to 38.95%. No significant change occurred in the erythrocytes of interme- diate age group (age 10–40 days). Time kinetics of changes in the proportion of old and young erythrocytes is shown in Figure 8B. These results suggest that the older erythrocytes in blood circulation may be most susceptible to the toxic effect of AF- SWCNTs. Figure 5. Effect of Control and acid functionalized SWCNTs on PS externalization in murine erythrocytes. Erythrocytes (0.56106 in 100 ml RPMI+1%FBS) from 6-8 weeks old C57BL/6 mice were incubated with given concentrations of control and acid functionalized SWCNTs for different time intervals at 37uC. PS externalization was studied staining with annexin V-PE antibody. Percent annexin V positive erythrocytes (mean 6 SEM of three values) have been shown. *p,0.05 as compared to control. doi:10.1371/journal.pone.0022032.g005 PLoS ONE | www.plosone.org 5 July 2011 | Volume 6 | Issue 7 | e22032