Effects of growth factors in neuronal viability and differentiation. To determine the requirement and effectiveness of different growth factors in neuronal survival and differentiation, we plated neurons with bFGF at DIV0 and changed complete media at DIV4. In the first experiment, at DIV4, different growth factors (bFGF, BDNF and GDNF) were added separately to the culture. A half-media change with the addition of respective growth factors was performed at DIV8, and the cells were fixed at DIV12. In the second experiment, cells received bFGF from DIV 0-8, thereafter media was aspirated off and fresh media containing bFGF, BDNF and GDNF were added separately. Cells were fixed at DIV 12. Fixed cells from both the experiments were permeabilized and then incubated with Pan-neuronal and GFAP or Pan-Neuronal and Nestin primary antibodies. Cells were incubated with appropriate fluorophore-conjugated secondary antibodies, washed off excess unbound antibodies, and immunofluorescent images were captured as stated in the text. Nuclei were stained by Hoechst dye. The figures were pseudocolorized using Adobe Photoshop CS4, where Pan-Neuronal-positive cells were colorized green, GFAP and Nestin-positive cells red and Hoechst were shown in blue. The figures depicted that cellular viability and differentiation were grossly decreased in the cultures that were supplemented by bFGF from DIV0-4, but received BDNF and GDNF from DIV4-12. However, cellular viability and differentiation were not affected in the group that received bFGF from DIV0-12. Contrarily, we have not noticed any alteration in cellular viability and differentiation in the cultures that were supplemented by bFGF from DIV0-8 and then supplemented by either bFGF, BDNF or GDNF from DIV8-12. These results indicate that supplementation by bFGF at the initial phase of the culture is essential for proper development and differentiation. Arrows indicate cell bodies that stain for both antibodies.