we described methods 
 in details elsewhere ; here we only provide an essential 
 summary of the methods used in 
 the present study and specifically point out some modifications . astrocytes 
 isolated from the visual cortices of 02-day - old c57bl/6 ( wild - type ) 
 or gfap ko mice were purified and maintained in cell culture as we 
 previously described . experimental protocols were approved 
 by the institutional animal care and use committee at the university 
 of alabama at birmingham . swcnt - peg solutes and 60 nm thick 
 swcnt - peg films were synthesized and characterized as we previously 
 described . swcnt - peg in this study contained 16.016.1 
 wt % peg . in experiments using peg solute , as a control for 5 g / ml 
 of swcnt - peg solute treated group , peg was added to the cells at 1 
 g / ml , i.e. , at the concentration corresponding to 20 wt % of 
 swcnt - peg . all imaging experiments were done at room temperature 
 ( 2225 
 c ) using a light microscope ( nikon te300 ) equipped with differential 
 interference contrast ( dic ) and epifluorescence illumination ( halogen 
 and xenon arc lamps , respectively ; 100 w ) . to acquire the images , 
 we used either a coolsnap - hq or a coolsnap - hq ( for supporting information figure s2 only ) cooled , 
 charge coupled device camera ( photometrics , tucson , az ) driven by 
 metamorph imaging software ver . all the raw fluorescence images had pixel intensities without saturation 
 and within the dynamic range of the camera ( 04095 for coolsnap - hq 
 or 016383 for coolsnap - hq ) . astrocytes were loaded 
 with -ala - lys - n-amca ( 20 m at 37 c 
 for 1 h in cell culture medium ) , and the coverslips containing the 
 cells were rinsed in the external solution , mounted onto an imaging 
 chamber filled with external solution , and examined live using a standard 
 dapi filter set and a 60 plan apo objective ( numerical aperture , 
 1.4 ) as described elsewhere . external solution contained 
 ( in mm ) : nacl ( 140 ) , kcl ( 5 ) , cacl2 ( 2 ) , mgcl2 ( 2 ) , d - glucose ( 5 ) , and hepes ( 10 ) ( ph 7.4 ) . alternatively , 
 -ala - lys - n-amca loaded astrocytes were subjected 
 to indirect immunocytochemistry . they were subsequently fixed and 
 labeled for gfap ( mouse monoclonal , 1:500 dilution ; icn cat . 69110 ; 
 mp biomedicals ; solon , oh ; followed by a tritc - conjugated secondary 
 antibody ) and analyzed for the cell area 
 by manual tracing ( based on the -ala - lys - n-amca images acquired using a standard dapi filter set ) , and for 
 the density , content , and occupancy of gfap - ir based on the images 
 acquired using a standard tritc filter set and a 60 plan apo 
 objective ( numerical aperture , 1.4 ) . to 
 test for the nonspecific binding of the secondary antibody and cellular 
 autofluorescence , parallel controls were run , in which the primary 
 antibody was omitted . briefly , all the images of 
 astrocytes ( exposed and not exposed to the primary antibody ) were 
 background subtracted ; background fluorescence was obtained from regions 
 of coverslips containing no cells . mean intensity plus six standard 
 deviations of the background subtracted images of astrocytes not exposed 
 to the primary antibody was used to calculate a threshold value for 
 gfap positive pixels . as in the previous work , only the astrocytes 
 that could be imaged within a single frame were analyzed in order 
 to avoid errors in the quantification of fluorescence intensity that 
 would otherwise occur during the stitching / autoleveling step in morphometric 
 analysis ( see below ) . the only difference in the current study is 
 that the coverslips ( thickness no . 1 ) instead of glass microscopic slides 
 and imaged through the coverglasses , to avoid assessment of any possible 
 errors associated with the absorption of light by the cnt films themselves 
 ( see figures s6 , s9 of ref ( 7 ) ) . to quantify astrocytic morphological changes , cells 
 were loaded 
 with calcein ( 1 g / ml calcein - am with 0.025% w / v pluronic acid , 
 invitrogen ; 15 min ) and , after de - esterification ( 15 min ) , imaged 
 live bathed in external solution ; a standard fitc filter set and a 
 60 plan apo objective ( numerical aperture , 1.4 ) were used for 
 the visualization of calcein fluorescence , as we described elsewhere 
 in detail . the area and 
 perimeter of the cells were obtained using a self - designed algorithm , 
 involving the stitching / autoleveling step when needed ( see figure 
 s1 of ref ( 4 ) ) , which 
 were further used to calculate the ff as we described elsewhere . in experiments assessing the adhesive and 
 proliferative characteristics 
 of astrocytes , nuclei of calcein - loaded astrocytes were labeled with 
 hoechst 33342 trihydrochloride trihydrate ( 5 g / ml , 15 min ; 
 invitrogen ) . imaging of live astrocytes was done using standard fitc 
 ( calcein ) and dapi ( hoechst ) filter sets and a 20 plan fluor 
 objective , 4 h and 4 days postplating . the number of nuclei per view 
 field ( 0.15 mm ) were counted as we described in detail 
 elsewhere . the cells positive for hoechst 
 and calcein were considered live , while the cells positive for hoechst 
 and negative for calcein were considered dead . statistical analysis 
 was done using the gb - stat v6.5 software ( dynamic 
 microsystems inc . , silver spring , md ) and sas software , version 9.2 
 of the sas software for windows ( sas institute inc . , the 
 number of subjects required for individual set of experiments was 
 estimated using power analysis ( set at 80% and  = 0.05 ) . since 
 some of the subgroups deviated from normality based on the shapirowilk 
 test for normality , we used nonparametric statistics . in all the experiments , 
 the mann  whitney u - test was used for the comparison between 
 wild - type and gfap ko astrocytes in each condition . to test the difference 
 between the 4 h and 4 day time points in the proliferation study , the two correlated groups were compared using the wilcoxon signed - rank 
 test ( figures 3 and 4 ) . for all the other experiments with comparison between the different 
 conditions in the same group , the multiple independent conditions 
 were analyzed using the kruskal wallis one - way anova ( kwa ) 
 followed by the dunn s or the newmankeuls test 
 to assess a statistical significance ( established at p < 0.05 ) in the effect induced by the cnt modalities when compared 
 to pei . the mann whitney u - test was used for the comparison 
 of morpho - functional properties between the untreated and peg treated 
 wild - type astrocytes ( supporting information , figure s2a  d ) ; the sole exception was the use of the student s t test for the assessment of the form factor .
alterations in glial fibrillary acidic 
 protein ( gfap ) levels accompany 
 the changes in the morphology and proliferation of astrocytes induced 
 by colloidal solutes and films of carbon nanotubes ( cnts ) . to determine 
 if gfap is required for the effects of cnts on astrocytes , we used 
 astrocytes isolated from gfap null mice . 
 we find that selected astrocytic 
 changes induced by cnts are mediated by gfap , i.e. , perimeter , shape , 
 and cell death for solutes , and proliferation for films .