Sorting of cells of the same size, shape, and cell cycle stage for a single cell level assay without staining

Abstract

Background: Single-cell level studies are being used increasingly to measure cell properties not directly observable in a cell population. High-performance data acquisition systems for such studies have, by necessity, developed in synchrony. However, improvements in sample purification techniques are also required to reveal new phenomena. Here we assessed a cell sorter as a sample-pretreatment tool for a single-cell level assay. A cell sorter is routinely used for selecting one type of cells from a heterogeneous mixture of cells using specific fluorescence labels. In this case, we wanted to select cells of exactly the same size, shape, and cell-cycle stage from a population, without using a specific fluorescence label.

Results: We used four light scatter parameters: the peak height and area of the forward scatter (FSheight and FSarea) and side scatter (SSheight and SSarea). The rat pheochromocytoma PC12 cell line, a neuronal cell line, was used for all experiments. The living cells concentrated in the high FSarea and middle SSheight/SSarea fractions. Single cells without cell clumps were concentrated in the low SS and middle FS fractions, and in the higher FSheight/FSarea and SSheight/SSarea fractions. The cell populations from these viable, single-cell-rich fractions were divided into twelve subfractions based on their FSarea-SSarea profiles, for more detailed analysis. We found that SSarea was proportional to the cell volume and the FSarea correlated with cell roundness and elongation, as well as with the level of DNA in the cell. To test the method and to characterize the basic properties of the isolated single cells, sorted cells were cultured in separate wells. The cells in all subfractions survived, proliferated and differentiated normally, suggesting that there was no serious damage. The smallest, roundest, and smoothest cells had the highest viability. There was no correlation between proliferation and differentiation. NGF increases cell viability but decreases the proliferative ability of the PC12 cells.

Conclusion: We demonstrated a pretreatment method to collect well-characterized, viable, single cells without using fluorescent labels and without significant damage to the cells. This method is quantitative, rapid, single-step, and yields cells of high purity, making it applicable for a variety of single-cell level analyses.

Figure 1

Living and dead cell fractions. A and D: two-dimensional profiles of the PC12 cells stained with calcein AM and PI. Calcein-PI (A) and SSarea-FSarea (D, left), FSarea-FSheight (D, middle) and SSarea-SSheight (D, right). Cells without dye (B) and cells permeabilized by 0.1 % saponin with dye added (C) were used to define the dead cells. Calcein-positive and PI-negative cells (living cells) are represented by blue dots and calcein-negative or PI-positive cells (dead cells) are represented by red dots.

Figure 2

Schematic representation of the particle structures, with peak height and area. A: Single cell with uniform content (a), single cell without uniform content (b), and doublet cells with uniform content (c). B: Time course of signals. C: Position of the cells in the height and area profiles. D: A sphere consists of uniformly dispersed small reflectors and has a central part with a slightly different refractive index (see Discussion).

Figure 3

Single and aggregate cell fractions. A, B: Number of aggregates in live cells, which were sorted according to their FS profile (A) and SS profile (B). A: Fifty gates in the FSarea-FSheight profile (left). Particles in each cross-section of gates a-c and gates 1–5 in the FSarea-FSheight profile were sorted. Then, the nuclei per particle were counted, and the number of cell aggregates per twenty-seven particles is shown (right). B: Eleven gates in the SSarea-SSheight profile (left), and the number of aggregates per twenty-seven particles (right). C: FSarea-FSheight (left), SSarea-SSheight (middle), and PI (right) profiles of the ethanol-fixed cells. The green and red dots shown in the SSarea-SSheight profile are the cells within the gate g and r shown in the FSarea-FSheight profile, respectively. Traces g, r and b in the PI profile correspond to gates g, r and b in the FSarea-FSheight profile, respectively. The numbers represent the percentage of cells in regions 1–4.

Figure 4

Living- and single-cell gates and the twelve subfractions. A: Living and single cell-rich gates in FSarea-FSheight (a, blue) and SSarea-SSheight (b, red) are shown. B: SSarea-FSarea profile. Left: dot plot of all cells (black) and cells in the a and b gates (purple dots). Right: contour plot of the cells in the a and b gates. The twelve sorting gates are represented by the green rectangles. C: Schematic representation of the twelve gates. SSarea and FSarea values were normalized to the smallest value and represented as SSn and FSn. D: The number of aggregate cells in the twelve subfractions (n = 120). The SYTO24-stained nuclei in the sorted cells were counted. Shading corresponds to that in C.

Figure 5

Cell size and shape of the sorted cells in the twelve subfractions. A: Phase-contrast micrographs of the sorted cells: spherical and small (a); spherical and large (b); oval (c); irregular surface (d). B-D: The diameter (d_a, B), elongation (e, C), and surface roughness (P/A, D) of the cells are shown. These values were evaluated from the phase-contrast images of cells (see Methods). Left, average for each subfraction; middle, average for SSn; right, average for FSn. Shading corresponds to that in Fig. 4C. The number of measured cells is shown on each column in B. The right ordinate of B is the relative cell volume (d_a³/d³), where d is the average of d_a at (SSn, FSn) = (1.00, 1.24) (arrow head). The relative cell volume at (SSn, FSn) = (2.00, 1.24) (arrow) was about two. Statistically significant for C and D at *P < 0.05; **P < 0.005; ***P < 0.0005. Statistically not significant for B and C at #P > 0.05.

Figure 6

Cell cycle of the sorted cells in the twelve subfractions. Live cells were sorted according to the twelve subfractions, then fixed and stained with PI. A: Two examples of PI profile. Upper trace: (SSn, FSn) = (2.33, 1. 48), lower trace: (1.33, 1.24). The left peak corresponds to the G1 phase and the right region corresponds to S, G2 and M phase. B: Percentage of S, G2 and M phase cells. Shading corresponds to that in Fig. 4C.

Figure 7

Viabilities of single-sorted cells plated in separate wells. Cell viability in each of the twelve sub-fractions with (B) and without (A) NGF. Left, average for each subfraction; middle, average for SSn; right, average for FSn. Shading corresponds to that in Fig. 4C. Statistically significant at *P < 0.05; **P < 0.005. C: SER versus NGF for each subfraction. The slope of the line is 1.