This study demonstrated a non-staining sample-pretreatment method to select well-characterized homogeneous cells from a cell line for single-cell level analysis. Although a cell line is genetically identical, it is morphologically heterogeneous due to aggregation, cell cycle, and phenotype variance. We used both peak height and area of light scattering parameters to sort PC12 cells into live single-cell fractions, without the use of a specific label. Although it is usually difficult to discriminate between single large cells and small cell aggregates, we successfully separated these classes using the height-to-area signal ratio of both FS and SS. The distinction methods were versatile enough to apply to both living and ethanol-fixed cells. Moreover, this discrimination method is applicable not only to pretreatment steps but also in the data acquisition stage.
We showed that the FSn is closely related to both the degree of cell elongation and the amount of DNA per cell, and that the SSn was proportional to the cell volume. This is in contrast to the commonly accepted association between light scattering and cell morphology that FS represents the size and SS represents internal structure (cf. owner manual of ALTRA). Light scattering profiles are closely related to morphological features including size, shape, surface detail, and intracellular structure of the particle [6, 9, 10, 12]. The dependency, however, varies according to the structure of the particles and the acquisition method applied [9, 13]. The commonly accepted association was based mainly on the analysis of different-size beads or different kinds of hemocyte using narrow angle analysis [9, 10]. However, current machines use wide angles; for example, the FS and SS of ALTRA are the summation of light scattering data from -23° to 23° and from 42° to 138° from the laser axis, respectively. Moreover, we tested an adherent cell line, in which the component cells are almost of the same structure and size. Thus, it is not surprising that our results differed from the accepted association. One possible explanation of our results is that the refractive index of the cell nuclei differs slightly from that of the cytosol and that the cell contains many small particles that reflect light and are distributed homogeneously (Fig. 2D). Diffraction and refraction are the main components of forward scatter and thus are affected by the size of the nucleus, which is roughly in proportion to the DNA content of the cell. On the other hand, reflection from small particles is the main component of side scatter. Because SSn or SSarea is the time integration of side scatter, it is proportional to the number of particles in the cell and as a consequence, is proportional to cell volume.
Light scattering can be used to gain quantitative criteria of cell appearance, which is a good indicator of cell properties. In experiments where data are acquired using a microscope, such as patch clamping, cells must be selected based on appearance to acquire meaningful data. Based on microscopic observation, cell aggregates, dead cells, and less vital cells can be excluded and cells from which data can be acquired, are selected. This empirical selection, however, may introduce an unknown bias to the experiments. Our single-cell assay showed that the roundest and smoothest cells have high viability. This intuitive result suggests that cell selection using light scattering data is consistent with empirical selection based on cell appearance, but without introducing unknown bias.
In the single-cell assay we used the auto-clone option. Because the conventional limiting dilution method is stochastic, there is no guarantee that individual cells are in separate wells. However, auto-clone enabled us to plate a precise number of cells directly into a separate well. Thus, this option would also be useful for single-cell assays of cell extracts, such as single-cell RT-PCR. This option also enabled us to assess cell viability (survival ratio) and cell proliferation independently, which is difficult to achieve in conventional mass culture. Finally, the single-living-cell assay results confirmed that the cell sorting method used in this study caused no significant damage to the cells, as the sorted single cells in the individual wells survived, proliferated and differentiated normally.