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Taking Cell Counting to the Next Level

A method using propidium iodide staining of the nuclei improves cell counting accuracy and safety.

by Ted Andrew
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Cell counting is a critical but often overlooked component of the consistency, health, and reproducibility of results in routine cell culture. Studies have shown that cell density can have pronounced effects on the phenotypic and genotypic response of the cells. These changes can cause wide swings in results in cell-based assays, susceptibility to compounds, and protein expression levels.

Manual cell counting with TBE

Many laboratories leave this critical step to the imprecise and time-consuming method of Trypan Blue Exclusion (TBE). Briefly, Trypan Blue is a dye that works by only penetrating cells with an impaired plasma membrane and being excluded from live, healthy cells. A volume of sample is then pipetted onto a hematocytometer and cell numbers are counted manually.


There are a number of issues associated with the TBE procedure:

• TBE also stains artifacts and cell debris

• TBE, as incubation time increases, will begin to permeate healthy cells

• Cells tend to aggregate, resulting in spurious results

• Sample volume is limited

• Counts are subjective and styles vary between researchers

• TBE is carcinogenic and pervasive


The TBE method is appropriate when a general phenotypic determination needs to made on every count along with the cell count. This determination is simply a sense of the general health of the cell. 


Automating the process

There are automated adaptations of the TBE method that eliminate the cell counting aspect of the procedure. However, these systems perform only as well as the algorithms that determine the live/dead differentiation in staining. In addition, they often require frequent calibration and service in order to maintain consistency in the readings. These systems can also be stymied by differences in cell size within the reading zone, and are still susceptible to aggregation, limited sample volume, and the increased incubation time. In fact, they offer only a marginal improvement and labor savings over the manual method.


Another method of counting cells is by using propidium iodide (PI) staining of the nuclei. PI intercalates with the DNA of the lysed cell. Using a device that has a fluorescence microscope and charged-coupled device (CCD) camera built-in, excitation occurs at 540nm and emission occurs at 600nm. The signal intensity is enhanced 20-fold when the PI is bound to the nucleus DNA, which enhances the signal-to-noise ratio. Since the system counts nuclei rather than cells, there is no calibration needed for cell size or morphology.


The NucleoCounter™ and cassette system offered by New Brunswick Scientific is one example of a PI system that can be used to obtain quick, repeatable, and accurate results. This system uses a unique cassette that consists of a calibrated plunger to ensure that volume is consistent between samples, drawing 50 μl of the treated cell suspension into a series of channels containing propidium iodide. These channels serve to further separate the cells as well as expose the nuclei to the PI. The cassette is then placed into a reader where the plunger is further depressed, automatically. This in turn draws the volume into a viewing window of calibrated volume. The window is read and an accurate count is made. The cassette is then simply discarded. This process means no messy dyes, no inaccurate pipetting, no calibration, and no flow cell to clean. The instrument is ready to go for the next sample.


Cell sample preparation is also quick and easy. The user pipettes a representative sample of culture from the cell suspension, adds an equal amount of lysis reagent and stabilization reagent, and aspirates with the plunger. The total sample preparation time and read is under one minute.


By utilizing this type of automated system, the researcher obtains more consistent cell numbers, resulting

in improved results for seeding, screening, and assay development. In addition, the results are then stored and are traceable for QC and statistical analysis.


An automated method of counting cells is the right choice where a consistent cell counting application is required. This would include any cell-based assay diagnostic protocol, any application requiring quality control in the cell-culture facility, and any protein expression protocol that requires batch-to-batch consistent results.


As cell culture becomes more sophisticated and the industry includes stem and insect cells as well, the traditional methods of cell counting will be held to a higher standard. In addition, data logging and results traceability are becoming more important in batch and GMP environments. An automated cell counting system is able to meet not only today’s needs but the needs of tomorrow’s laboratory as well.