Counting and checking the viability of cells in culture is an essential part of good cell culture practice to ensure that cells are healthy and seeded at an appropriate density into medium when they are being resuscitated, passaged or even cryopreserved. In the European Collection of Authenticated Cell Cultures (ECACC) laboratories we perform many cell counts every day as we grow and maintain the thousands of cell lines that are held within the collection, and our cell culture scientists have a great deal of experience of counting cells from many different species and tissue types.
Use of a haemocytometer and trypan blue stain to count and assess the viability of cells in culture has been the method of choice for researchers for many years because it is cheap, fairly quick to perform and is applicable to many cell types. This method does, however, require a certain degree of skill and results obtained can vary with different operators. Dilution of cells is often required in order to make counting cells feasible and this dilution step can make the counts less accurate. Novices may also find the calculations associated with this method to be problematical. A simple protocol for cell counting using a haemocytometer can be found here.
For those of us performing frequent cell counts, using a haemocytometer can be tedious and different methods for automated counting of cells have been developed over the years. Automated cell counters are now becoming more affordable, increasingly popular and offer many benefits. They are generally easy to use, reduce the time taken to produce a cell count and give more reliable and reproducible results. Although they are very useful for most commonly encountered cell types users should be aware of the limitations such as may become apparent when trying to count unusually shaped cells such as neurons, mixtures of cell types, very small cells or clumped cells.
Several main technologies have been applied to counting cells in culture:
Cells suspended in an electrically conductive medium are sucked through a small aperture and as each cell passes through the aperture it causes a brief change to the electrical resistance of the liquid. The magnitude of the change allows the size of the cell and the concentration of cells to be determined. There are many types of cell counter available based on the coulter counter principle, varying in sensitivity, solvent compatibility, speed of measurement, sample volume, dynamic range and reliability.
Flow cytometers are relatively expensive and generally used for more sophisticated analyses than cell counting such as looking at cell surface markers or for sorting cells. The basic principle is that cells in suspension in a liquid flow, passing through a beam of light, will scatter or absorb the light and produce fluorescence which can be detected. The pattern of scattering or absorption can also be used to determine the size and shape of cells which can then be sorted by electrostatic deflection based on their characteristics if required.
In recent years microscopes which can capture digital images of cells as they grow in an incubator have been developed. These digital images can be manipulated and analysed by a variety of software to count cells per unit area in the flask or in combination with fluorescent antibodies and other techniques.
A variation on image analysis is the use of a DNA stain to identify nuclei in cells which can then be counted. Unlike other cell quantification methods, automated nuclei counters eliminate manual counting and do not rely on the retention of physical and/or morphological properties of the cells.
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