Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and accuracy. 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, 71% of 400 researchers surveyed1 who count cells use a hemocytometer to do so. While hemocytometry is inexpensive, it is laborious and subject to user bias and misuse, which 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 errors 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 rate2.
To alleviate the tedium associated with manual counting, 29% of researchers count cells using automated cell counting devices; these include vision-based counters, systems that detect cells using the Coulter principle, or flow cytometry1. For most researchers, the main barrier to using an automated system is the price associated with these large benchtop instruments1.
With its Scepter cell counter (Figure 1), Millipore has captured the ease of automated instrumentation and accuracy of Coulter counting in an affordable, handheld format. The system employs the Coulter principle of impedance-based particle detection 3 in a miniaturized format enabling rapid cell counts. The instrumentation has been collapsed into a device the size of a pipette and uses a combination of analog and digital hardware for sensing, signal processing, data storage, and graphical display. The disposable tip is engineered with a microfabricated, cell-sensing zone that enables discrimination by cell size and cell volume at sub-micron and sub-picoliter resolution. Enhanced with precision liquid-handling channels and electronics, the Scepter cell counter provides cell population statistics graphically displayed as a histogram.
Operation of the Scepter cell counter is similar to using a pipette such as that found in most laboratories.
Prepare the sample:
A single-cell suspension, diluted with phosphate buffered saline (PBS) for sufficient conductivity to a concentration of 10,000-500,000 cells/mL (operating range), is optimal for obtaining an accurate cell count – EmbryoMax® 1x PBS (Millipore) is recommended. A sample volume of 100 µL in a 1.5 mL microcentrifuge tube is recommended. Other tubes may not be able to accommodate the width of the tip or provide sufficient sample depth for the instrument to function properly.
Perform cell count:
The Scepter cell counter is turned on by depressing and holding the toggle on the back of the instrument. Once on, the instrument will prompt the user to attach a tip. After the tip is attached, the Scepter unit displays 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, then calculates cell concentration and displays a histogram of cell size or cell diameter on its screen.
Analyze the data:
The upper and lower limits of the histogram, called gates, are set either automatically based on the histogram profile, or are set to the same gates used in the previous count. After the count is complete and the histogram is displayed on the instrument, the gates can be moved manually to fine-tune the data. Up to 72 histograms can be stored on the instrument itself and, if desired, uploaded and saved to a personal computer with the included software.
Linearity and precision of Scepter cell counter across multiple cell lines
The Scepter cell counter was used to count cell suspensions from 19 different cell lines (Table 2). Cells were harvested, diluted in EmbryoMax PBS, and counted using a Coulter Counter® to determine the theoretical starting concentrations. The original cell concentration was divided by the fold dilution at each serial dilution step to determine theoretical concentrations listed in Table 3. The high degree of linearity as shown by the R2 values indicates that Scepter counting is a reliable method for the cell lines tested, across a wide linear operating range (Figure 4).
Table 1: Scepter Device Specifications
The precision of Scepter cell counting was determined by calculating coefficients of variation at each data point, for each cell line individually, as represented by the mesenchymal stem cell line data in Table 3. The overall relative accuracy of Scepter counting is shown in Figure 5, by comparing counts obtained from Scepter counting with counts obtained from Coulter counting.
Linearity and precision of Scepter counting compared to other systems
Counts of each cell line in Table 2 were performed using a Z2™ Coulter Counter (Beckman Coulter), the Scepter cell 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.
Results show that cell concentrations measured by Scepter counting closely match theoretical cell concentrations with high linearity (Figure 6). Scepter counting is more precise than vision-based counting and hemocytometry, displaying smaller standard deviations (Figure 6) and smaller average coefficients of variation (Figure 7).
The time required to perform cell counts using each of the systems described in Figure 6 was compared using a 500,000 cells/mL sample (Figure 8). Scepter counting (14 seconds on average, under these conditions) is significantly faster than any of the other cell counting methods. Most dramatically, it is ten times faster than hemocytometry.
Comparing the performance of the Scepter cell counter to results from other counting methods, we conclude that this new handheld, automated cell counter delivers precise, fast, and reliable cell counts over a wide operating range. 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.
To continue reading please sign in or create an account.Don't Have An Account?