Fig. 5

Phenotypical characterization of HMC3 cells under basal conditions and in response to IFNγ. HMC3 cells were plated at the density of 30,000 cells/cm² in T25 flasks, and grown for 3 days when cells were almost confluent. In the experiments in which INFγ was used, cells were stimulated for the last 36 h. Controls did not receive any stimulus for the same time period (“resting” HMC3 cells). For detection of intracellular antigens, aliquots of 1 × 10⁶ cells in 100 μl were fixed using BD cytofix (BD Pharmingen) at 4 °C for 30 min, and permeabilized with BD FACS permeabilizing solution at 4 °C for 30 min. Cells were then stained using the following antibodies: PE-conjugated anti Human GFAP mouse Mab, BD Bioscience (a), and PE-CF594-conjugated anti Human CD68 mouse Mab, BD Bioscience (e), according to the manufacturer’s instructions. For the evaluation of surface antigens, aliquots of 5 × 10⁵ cells in 100 μl were directly incubated in PBS buffer containing the following antibodies: PE-CF594-conjugated anti Human HLA-DR mouse Mab, BD Bioscience (b); FITC-conjugated anti Human CD14 mouse Mab, BD Pharmingen (c); and PE-conjugated anti Human CD11b mouse Mab, E-Bioscience (d). Cells were analyzed by the 6-parameter (2 scatter and 4 fluorescence signals) Coulter Epics XL flow cytometer (Beckman-Coulter). Control histograms (white histograms) indicate level of cell autofluorescence in the emission wavelength that pertains the fluorochrome-conjugated Mab. Panel (a) shows expression of GFAP on HMC3 cells at passage 4 (dark gray), and on the human glioblastoma U373 cell line (light gray) that constitutively expresses GFAP. As autofluorescence signal in the two cell lines was similar, only one representative histogram is plotted in panel (a) (white histogram). As shown in this panel, the HMC3 cells stain negatively for GFAP (the dark gray histogram completely overlaps the background histogram), whereas the U373 cells express the target antigen. Two different populations expressing GFAP at different levels were identified (light gray plot): 68% of the U373 cells express GFAP at low level (GFAP-dim), whereas 32% of the cells express GFAP at higher level (GFAP-high). Panels (b–e) show results from HMC3 cells obtained at passage 7. In these panels, white histograms indicate cell autofluorescence, light gray histograms and dark gray histograms are representative of control (“resting”) HMC3 cells and IFNγ-treated HMC3 cells, respectively. Gating strategy: all analyses were obtained after gating according to morphological characteristics (not shown). As levels of autofluorescence did not change according to cell treatment, one representative example is plotted for each emission wavelength. Linear regions are used for calculating percentage of positive cells (plots A–B). Central tendency of CD68 expression on HMC3 cells in the different experimental conditions is represented by the mean fluorescence intensity (MFI, see text)