Introduction

Counting cells is often a necessary but tedious step for in vitro cell culture. Cell counts are important for monitoring cell health and proliferation rate, assessing immortalization or transformation, seeding cells for subsequent experiments, transfection or infection, and preparing for cell-based assays. It is important that cell counts be accurate, consistent, and fast, particularly for quantitative measurements of cellular responses.

Despite this need for speed and accuracy in cell counting, a survey (1) of 400 researchers who count cells revealed that 71% use a hemocytometer. While hemocytometry is inexpensive, it is laborious and subject to user bias and misuse that results in inaccurate counts. Hemocytometers are made of special optical glass on which cell suspensions are loaded in specified volumes and counted under a microscope. Sources of error in hemocytometry include uneven cell distribution in the sample; too many or too few cells in the sample; subjective decisions as to whether a given cell falls within the defined counting area; contamination of the hemocytometer; user-to-user variation; and variation of hemocytometer filling rate (2).

To alleviate the tedium associated with manual counting, 29% of the researchers in the survey count cells using automated cell counting devices; these include vision-based counters—systems that detect cells using the Coulter principle—and flow cytometers (1). For most researchers, the main barrier to using an automated system is the price associated with these large benchtop instruments (1).

The Scepter™ cell counter (Millipore) combines the ease of automated instrumentation and the accuracy of Coulter impedance-based particle detection (3) in an affordable, handheld format (Figure 1). The instrument, which is the size of a pipet, uses a combination of analog and digital hardware for sensing, signal processing, data storage, and graphical display. The disposable tip (Figure 1, inset) has a microfabricated, cell-sensing zone that can discriminate cell size and cell volume at sub-micron and sub-picoliter resolution. Enhanced with precision liquid-handling channels and electronics, the Scepter cell counter graphically displays cell population statistics as histograms. Counting is typically completed in less than 20 seconds.

Figure 1. Parts and functions of the Scepter handheld automated cell counter, including the cell-sensing orifice and the precision-engineered microchannel. (Click to enlarge)

Materials and methods

Operation of the Scepter cell counter is similar to using a pipet such as that found in most laboratories.

Cell lines tested

Nineteen lines, which cover the range of cell types used in most cell research laboratories, were used to validate performance of the Scepter cell counter. The cell lines represent adherent, suspension, differentiated, pluripotent, normal, and transformed cells, including HeLa (Millipore, Cat. no. 20-385), K562 (ATCC, Cat. no. CCL243), U266 (ATCC, Cat. no. TIB-196), Cos-7 (Genlantis, Cat. no. C505100), and human mesechymal stem cells (Millipore, Cat. no. SCR108).

Sample preparation

Single-cell suspensions were diluted with phosphate-buffered saline (1× EmbryoMax® PBS; Millipore) for sufficient conductivity and counted using a Z2™ Coulter Counter® (Beckman Coulter). Dilution series were prepared in 1.5-mL microcentrifuge tubes. The dilutions ranged from 10,000 to 500,000 cells/mL (Scepter operating range) with a minimum sample volume of 100 µL. The starting cell cof concentration was divided by the fold dilution at each serial dilution step to determine theoretical cell concentrations. Four replicates of each dilution were prepared for Scepter counting.

Scepter cell counting

The Scepter cell counter was used to count cell samples by following the detailed on-screen instructions for each step of the counting process. Briefly, the user depresses the plunger, submerges the tip into the solution, then releases the plunger to draw 50 μL of cell suspension into the tip. The Scepter cell counter detects each cell passing through the tip's orifice, calculates the cell concentration, and displays a histogram of cell size or cell diameter on its screen.

Cell counting by other methods

Counts of each cell line were also performed using the Coulter Counter, an automated vision-based counter, such as Vi-CELL® (Beckman Coulter) or Countess™ (Life Technologies) system, and a hemocytometer. Counts were performed according to manufacturer's instructions using the same cell starting suspension and identical dilutions.

Data analysis

Automated gating (shown as red vertical lines in Figure 2) was applied to each histogram to exclude cell debris from cell concentration calculations. Histograms were uploaded to a personal computer using the included software and USB cable. Meta-analyses were conducted by exporting the data to Microsoft Excel®.

Figure 2. The Scepter cell counter's screen displays histogram data on cell populations, distributed by either diameter (left) or volume (right). (Click to enlarge)

Results

Linearity and relative accuracy of the Scepter cell counter across multiple cell lines

The Scepter cell counter was used to count multiple cell lines with diverse physical characteristics. Scepter counting exhibited a high degree of linearity as shown by the R² values (R² > 0.993) for the cell lines tested across a wide linear operating range (Figure 3). The data also indicate high relative accuracy, as evidenced by the close agreement with theoretical cell concentrations.

Figure 3. Cell concentrations determined by Scepter counting compared to theoretical cell concentrations for five representative cell lines (out of 19 total cell lines tested), over the Scepter cell counter's operating range. (Click to enlarge)

Linearity and precision of the Scepter cell counter compared to other cell counting systems

Cell concentrations measured by Scepter counting matched theoretical cell concentrations more closely than measurements using hemocytometry or automated vision-based counting (Figure 4). Scepter counting was also more linear and precise than automated vision-based counting and hemocytometry, displaying smaller standard deviations (Figure 4). The percent coefficient of variation (%CV) was calculated for each dilution of each cell line for each counting system, and the average %CV across all 19 cell lines was recorded as a measure of precision. Scepter counting displayed smaller average %CVs compared to automated vision-based counting and hemocytometry across all tested cell lines (Figure 5).

Figure 4. COS7 cell concentrations measured using various systems. (Click to enlarge)

Figure 5. Shown are the average %CVs (over all 19 cell lines) with respect to cell concentration and counting method. (Click to enlarge)

Discussion

Comparing the performance of the Scepter cell counter to results from other counting methods, we conclude this new handheld, automated cell counter delivers precise and reliable cell counts over a wide operating range. Scepter counting is compatible with multiple cell types with diverse growth characteristics, diameter, and differentiation status.

The superior functionality of Scepter counting is likely a result of the precision-engineered technology embedded into the tip and the sophisticated counting instrumentation based upon the Coulter principle. This performance quality, combined with the Scepter cell counter's convenient, intuitive form, suggests that Scepter counting will be quickly integrated into the workflow of cell culturists wishing to improve reproducibility of cell-based assays and alleviate the pain of rudimentary cell counting.