Characterization of the TRH/GFP+ cell population in primary cultures of rat fetal hypothalamic cells. A) Strategy to obtain a neuronal population enriched in TRH cells from primary cultures of fetal (E17) hypothalamic cells. Primary cultures were transfected with a vector driving GFP expression under the control of the Trh promoter (TRH-GFP; referred as phrTRH-GFP in materials and methods) and GFP+ cells were sorted by flow cytometry. GFP+ cells were used to generate the target cRNA to hybridize the U34A array. Non-sorted hypothalamic cells and non-transfected cells were used as control. B) Representative FACS plots indicating the number of GFP+ cells (M1) in a logarithmic scale for 1 × 104 events. The upper panel indicates the distribution of fluorescence in the non-transfected cell population; the middle panel indicates the distribution of fluorescence in cells transfected with phrTRH-GFP; the percentage of GFP+ cells is indicated on the top. Lower panel: after GFP+ cells were isolated by preparative FACS, they were submitted to a second pass through the FACS to determine the percentage of GFP+ cells in the enriched population; it rose to 94%. C) Several marker transcripts were amplified by RT-PCR. The thyrotropin releasing hormone (trh) and tau transcripts were used as neuronal markers, the glial fibrillary acidic protein (gfap) as glial cell marker and the green fluorescent protein (gfp) for transfection control. The glyceraldehyde 3-phosphate dehydrogenase (g3pdh) was used as a control. D) Analysis of transcripts identified in the GFP+ cell population according to microarray data: neurofilament heavy polypeptide (nefh), vitamin D3 up-regulated transcript (vdup1), and krüppel-like factor 4 (Klf4); the g3pdh was used as internal control. GFP+, transfected purified cells; GFP+/-, transfected non-purified cell population; NT, non-transfected cell population.