Separation of cell subpopulations from embryonic chick neural retina with fluorescence-activated cell sorting

Fluorescence-activated cell analysis and sorting was used to characterize and separate cell subpopulations from the developing chick retina according to size, stage in the cell cycle and location in the tissue. It was shown that uptake of rhodamine B isothiocyanate (RITC) protein stain, light-scattering at large angles, and the time of flight of cells through the laser beam all correlate with cell size. Vital staining of DNA by H33342 permits cell separation according to phase in the cell cycle (G1, S, G2 + M). Further, a method was developed for separating postmitotic cells, based upon the quenching of H33342 fluorescence by the incorporation of 5-bromo-2'-deoxyuridine (BrdU) into DNA. Quantitative data reflect the decrease of DNA-synthesizing cells and the increase of postmitotic cells as the development of the retina proceeds. Isolation of cells on the basis of their location in the whole tissue is achieved by exposing the desired surface (e.g. the inner (concave) surface of the retina, which is rich in ganglion cells) to a suspension of Fluorescamine, which stains only cell layers close to the surface. Sorting out the fluorescing cells yields a high proportion of cells with the appearance, size and postmitotic characteristics of ganglion cells.