Data on retinoic acid and reduced serum concentration induced differentiation of Neuro-2a neuroblastoma cells

The present data describe the relative neuro-2a cellular differentiation induced by reducing serum concentration (0.1% FBS) in DMEM in the presence/absence of 20 μM retinoic acid (RA). Neurite outgrowth was observed within 24 h in DMEM supplemented with reduced serum and retinoic acid (GpIV). The CFSE based proliferation assay data signified cessation of neuro-2a cellular proliferation in GpIV. An increase in the number of cells arrested at G0/G1 phase was also evident in GpIV and DMEM supplemented with 0.1% FBS (GpIII). Moreover, GpIV cells had improved mRNA and protein expression of Rbfox3/NeuN and choline acetyltransferase (ChAT).


Value of the data
The present data describe the cell culture conditions, driving reproducible neuro-2a cellular differentiation within 24 h and will be useful for people working in this domain.
The data describe a stepwise strategy to be used for characterizing neuro-2a cellular differentiation.
The present data demonstrated induced expression of ChAT that can be helpful for researchers in their experimental planning.
Phase contrast microscopy ( Fig. 1-A-D) was used to visualize the morphological changes (neurite extension) induced by various treatment conditions. Extensive increase (P o 0.001) in the neurites length ( Fig. 1E) and branching pattern ( Fig. 1F) was seen in GpIV at 24 h, whereas GpIII did show moderate neurite extension. CFSE assay was performed to assess the rate of cellular proliferation and data for the same are plotted as bar graphs ( Fig. 2-E, F). The difference in the mean fluorescence intensity between GpIV and other groups was observed, which indicated the low rate of proliferation in GpIV. Additionally, it has been observed in previous reports that RA induces cell cycle arrest at the G 0 /G 1 phase [1]. In order to delineate this type of response, cell cycle analysis was performed (

Neuro-2a cell culture and differentiation
Neuro-2a cells were procured from NCCS Pune, India and maintained in DMEM (Sigma, D7777) supplemented with 10% FBS (Thermo Fisher Scientific, 10270106) at 37°C, 5% CO 2 conditions. For differentiation, the cells were plated at a density of 2 Â 10 4 cells/cm 2 and maintained in the standard growth media for 24 h. Next day, the standard growth media were replaced with the differentiation media of various compositions (Table 1) in their respective wells and incubation was continued for the next 24 h. Retinoic acid (R2625) was procured from Sigma-Aldrich.

Neurite length measurement
Neuro-2a cells were grown in 6-well multi-dishes (Thermo Fischer Scientific, 140675) and differentiated as per above method. Images were captured with the Nikon Eclipse TS100 at 20 Â magnification using VEZU US300. Neurite length was measured using ImageJ-Simple Neurite Tracer plugin [2]. Briefly, Images were first converted to 8-bit luminance format for analysis by the plugin. For defining neurite's path, a manual tracking was done from the junction of the cell body and neurite up to the neurite tip. Only neurites 4 1 μM in length were taken for final analysis. Branching patterning was further assessed using the Sholl analysis function in the same plugin. The neurite's path was manually defined, followed by assigning soma as a center of analysis. Using plugin parameters (Standard axis, Step radius ¼ 0 μM, Enclosing radius cut off ¼ 1, Sholl methods ¼ Linear, Polynomial  Values are mean 7 S.E. P r 0.05 (α, β, γ), P r 0.01 (αα, ββ, γγ), P r 0.001 (ααα, βββ, γγγ) were considered to be statistically significant. α, Compared to GpI; β, Compared to GpII; γ, Compared to GpIII. Additionally, mean values, S.E., ANOVA, and Tukey's multiple comparisons statistics (post hoc) are shown in tabular form below their respective graphs.

Statistical analysis
Values are presented as the mean 7S.E. Statistical analysis was done with SigmaPlot 11.0 with one way ANOVA and post hoc analysis using Tukey's multiple comparison test. P r 0.05, P r 0.01, P r 0.001 were considered to be statistically significant.