Fluorescence activated cell sorter facs

TauGFP-positive and negative E14.5 fetuses, from crosses to outbred CD-1 strain mice, were dissected in ice-cold Earle’s Balanced Salt Solution (EBSS), pre-equilibrated in 5% CO₂. Whole brains, hippocampus, cerebral cortex, ventral telencephalon, central and dorsal thalamus, and midbrain and hindbrain tissues were dissected and dissociated using the Worthington Papain Dissociation System, (Worthington Biochemical Corporation, USA). Dissociated cells were sorted for green fluorescence using a Fluorescence Activated Cell Sorter (FACS; Becton Dickinson, Rutherford, NJ, USA) to generate histograms of green fluorescence intensity (FL1 channel) versus cell number for 10,000 cells per sample. Gates identifying cell populations with different fluorescent intensities were defined using a tauGFP-positive TP6.3 brain sample, considered to contain 100% fluorescent cells and the same gates were applied to all samples. After gating to exclude outliers (predominantly dead cells), the percentage of cells in three gated regions, corresponding to different fluorescent intensities, were compared: region M1 included all fluorescent cells, M2 included cells with low fluorescence and M3 included cells with high fluorescence.

Flow cytometry analysis of Propidium Iodide (PI) stained nuclei was done to assess the effect of epigenetic drugs on the cell cycle distribution. AZA (15 μM), SAM (15 μM), TSA (100 nM), and SFN (10 μM) treated cells were incubated in respective media with 5% FBS for 24 h. The cells were then trypsinized, collected by centrifugation (500 × g for 5 mins at 4°C), washed twice with PBS and then fixed in 90% ice-cold methanol. After incubation at -20°C for 1 h, cells were centrifuged and resuspended in PBS followed by treatment with RNaseA (500 U/mL) to digest the residual RNAs and stained with PI (10 μg/mL). Samples were incubated for 30 min at 4°C and cell cycle analysis was performed with a Becton-Dickinson fluorescence-activated cell sorter (FACS).