Manual cell counting V.S. Automated cell counting
How can automated cell counters overcome the 4 major problems with manual cell counting?
Manual cell counting is still the golden standard method of cell counting in many labs. But, as presented in this article, there are several issues with the results obtained by manually counting cells with trypan blue and hemocytometer.
Cell counting with image cytometry and the automated cell counters provides a solution to all of these problems.
Furthermore, methods for counting adherent and aggregated cells are also developed for the NucleoCounter® system, allowing for fast and specific measurements of cells that are normally challenging to estimate specific cell concentrations of. This provides an immense advance in precision and reproducibility for mammalian cell culturing.
Challenges in manual cell counting of mammalian cells
The time at the microscope counting cells is both laborious and time-consuming. As mammalian cells are delicate systems, they require high reproducibility of setup during culturing. It is vital to know the specific cell concentration and viability of a cell sample to obtain reproducibility in subculturing, monitoring growth rates or for cryopreservation1,2. But manual cell counting of the cell concentration can give rise to large errors in cell concentration and viability.
The 4 biggest problems with manual cell counting:
- Human perception of definition of cell
- Volume, dilution and pipetting errors
- Trypan blue exclusion for viability determination
- Counting enough events
1. Human perception and definition of cells
The manual recognition of when a cell is a cell or when it is cell debris or other particles can be challenging even for the trained eye. Each person performing the manual cell count has a certain set of criteria that defines a cell and the threshold of brightness of the stain in order to count it as viable or dead. These interpersonal differences in manual cell recognition can be crucial for the experimental setup. If multiple users count the same sample it is not uncommon to see a variance significantly higher than the mean of a Poisson distribution3.
Figure 1. Cell samples with cell debris are very challenging to count correctly manually. In comparison, fluorescent events are clearly visible.
2. Volume, dilution and pipetting errors with manual cell counting
3. Viability determinations: Trypan blue or DAPI?
4. Counting enough cells
Avoid human interference using the Via1-cassette™
The Via1- Cassette™ (Fig. 3) is designed to overcome all human interference in cell counting:
- No human bias will influence the result – the definition of cells are performed with the software based on acridine orange signal, and will be the same in every analysis.
- No user or pipetting errors interfere with data generation – the cassettes have a pre-calibrated volume of the measurement chamber giving a precise and reproducible result.
- The cassettes are loaded with immobilized acridine orange and DAPI for definition of total cell and dead cell population, respectively.
- The instrument will tell you if you have too few or too many cells in the sample.
The cassettes are disposable plastic units that contain the fluorescent dye used for analysis. A cell sample is easily loaded into the cassette by submerging the inbuilt pipette into the cell suspension and pressing the piston.
Automated cell counters for higher precision and reproducibility
Fluorophores for cell counting
- Phelan, M.C., Basic techniques in mammalian cell tissue culture. Curr Protoc Cell Biol, 2007. Chapter 1: p. Unit 1.1.
- Freshney, R.I., Basic Principles of Cell Culture. 2006: p. 1–22.
- Nielson, L., G. Smyth, and P. Greenfield, Hemacytometer Cell Count Distributions: Implications of Non-Poisson Behavior. Biotechnology Progress, 1991. 7(6): p. 560-563.
- Shah, D., et al., NucleoCounter-An efficient technique for the determination of cell number and viability in animal cell culture processes. Cytotechnology, 2006. 51(1): p. 39-44.
- Tennant, J.R., Evaluation of the Trypan Blue Technique for Determination of Cell Viability. Transplantation, 1964. 2: p. 685-94. PMID: 14224649.
- Robbins, E. and P.I. Marcus, Dynamics of Acridine Orange-Cell Interaction. I. Interrelationships of acridine orange particles and cytoplasmic reddening. J Cell Biol, 1963. 18: p. 237-50. PMID: 14079487.
- Kubista, M., B. Akerman, and B. Nordén, Characterization of interaction between DNA and 4′,6-diamidino-2-phenylindole by optical spectroscopy. Biochemistry, 1987. 26(14): p. 4545-53. PMID: 3663606.
Merethe Mørch Frøsig, PhD, MSc
Field Application Scientist, ChemoMetec A/S
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