200 inverted fluorescent microscope

The self-renewal and differentiation markers of the NPCs were also assessed by immunofluorescence34 (link). For immunofluorescence staining analysis cells were incubated with the primary antibody Nestin (1:400; MAB353, Millipore), Tuj1 (1:500,05-549, Upstate), Sox2 (1:100; 481400, Life technologies), Map2(1:400; M1406, Sigma), Vimentin (1:100; V6630, Sigma), GFAP (1:500; MAB360, millipore) overnight at 4 °C. The secondary antibodies are anti-mouse IgG FITC antibody (1:200, St. Louis, MO, USA) and anti-rabbit IgG FITC antibody (1:1000, St. Louis, MO, USA) diluted in blocking buffer. Nuclei are counter-stained with Hochest 33342 (1:500; 94403, St. Louis, MO, USA). The fluorescent images of 2-D cultured cells were visualized on a Zeiss 200 inverted fluorescent microscope (Carl Zeiss, Jena, Germany). The number of immunostained cells was counted in each of three random fields per well and the fluorescence images were selected randomly. The quantification of the immunofluorescence signal was performed by Image-Pro Plus software (Media Cybernetics, Bethesda, MD). The fluorescent images of 3-D cultured cells were taken with a Leica TCS SP5 scanning laser confocal fluorescence microscope (Leica Microsystems, Inc., Germany).

Fluorescein diacetate (FDA) can be used to assess the cell viability of numerous cell types, as it generates observable differences in the fluorescence produced by live versus dead cells [22 (link)]. The collagen scaffold with implanted cells was incubated with 100 g/ml FDA for 1 min in the dark and then rinsed with phosphate-buffered saline. Samples were observed using the Zeiss 200 inverted fluorescent microscope (Carl Zeiss, Jena, Germany).

The combined use of the fluorescein diacetate (FDA) and propidium iodide (PI) is one of the most common fluorescence-based methods to assess the viability of cells. The principle of such staining is that PI can only cross membranes of non-viable cells whereas FDA is metabolized by viable cells, which leads to fluorescein fluorescence. Cells were incubated with this staining solution for 4–5 min at room temperature in the dark, and then the staining solution was removed and phosphate buffered solution (PBS) was added. The staining solution contained 100 μg/ml FDA (5 mg/mL in acetone; Sigma Chemical, St. Louis, MO, United States) and 60 μg/ml PI (2 mg/mL in PBS; Calbiochem). Samples were analyzed using the Zeiss 200 inverted fluorescent microscope (Carl Zeiss, Jena, Germany).