Cell counting method：From counting board to fully automatic cell counter

Many common experiments and tests require scientists to first accurately calculate the number or density of cells. These operations include simple cell culture maintenance (such as cell passaging) and quantitative experiments (such as qPCR). Cell counting can be accomplished through various methods, and this article will comprehensively introduce these methods and their advantages and disadvantages.

1 Counting board/blood cellscountdevice

Using blood cellscountdeviceFirst, clean with 70% alcohol and lens papercountdeviceThen gently place the cover glass on the counting board. If the cover glass is placed correctly, the Newton's ring phenomenon (concentric circular colored pattern) can be observed. Take a small portion of the cell suspension using a pipette, place the pipette on the edge of the counting plate, and allow the suspension to flow into the counting chamber through capillary action. If cell viability needs to be tested, trypan blue should be mixed into the cell suspension in a 1:1 ratio before being added to the counting chamber.

blood corpusclecountdeviceIt is an early method specifically used to obtain accurate cell counts, with special grids on the counting plate. Usually, the large grid is 1 millimeter by 1 millimeter and further divided into small grids of 0.05 millimeters by 0.05 millimeters. The edge of the counting room is designed to fix the cover glass 0.1 millimeters above the marking grid, forming a known volume area.

After focusing the microscope on an area of the counting chamber, useCounting deviceCount the cells. Usually, a 1 square millimeter (100 nanoliters) area is used and counting is done using a 4x or 10x objective lens, but the specific choice depends on cell size and density in the suspension. This process is usually repeated in four different 1 square millimeter areas, and the result is taken as the average. If detecting cell activity, live cells and dead cells should be counted separately, where dead cells are stained blue by trypan blue due to membrane damage.

Figure 1 BloodcellSchematic diagram of counting board grid

pros and cons

Manual counting is a budget saving method, but it is also the most time-consuming and tedious. Due to human error, especially when counting multiple times continuously, the reliability is low. In addition, frequent counting may also lead to eye fatigue. If the laboratory only needs occasional counting and does not require high precision, then blood cellsCounting deviceIt's a good choice. But this method is insufficient when frequent counting, improved accuracy, or high-throughput applications are needed. In addition, manual counting is difficult to distinguish between different types of cells in suspension unless there are significant differences in size or shape among cell types.

2 Automated cellsCounting device

Automated cellsCounting deviceIt is a fast, simple, and automated alternative to manual counting. They are based on blood cellsCounting deviceThe same principle is used to complete the operation by counting cells multiple times within a known area and taking the average. These devices can also distinguish between live and dead cells using dye exclusion methods such as trypan blue. Automated cellsCounting deviceIt can be a standalone device or connected to a computer for use. mostCounting deviceIn addition to providing cell counting, it can also count information such as cell size.

Fluorescent cell countingceremony(For example, Luna FL) ™ Double fluorescence cell countingceremony）Distinguish between live and dead cells and perform cell counting using common staining agents such as acridine orange and propidium iodide. These types of cell countsceremonyIt also performs well in counting cells in cultures that may be contaminated by non cellular debris (such as primary cells), as dyes can clearly distinguish cells.

pros and cons

automaticCounting devicesuitableGeneralCell counting and activity detection. It is easy to operate, cost-effective, and greatly reduces the labor required for manual counting. automaticCounting deviceAccurate and reliable, but there may be challenges when counting irregularly shaped, very small, extremely thin suspensions, or containing multiple cells that need to be distinguished. For most cell types and applications, automaticCounting deviceProvides efficient counting performance.

3 CoulterCounting device

CoulterCounting deviceIt is not an optical instrument, but counting cells by measuring the resistance changes of microchannels. The resistance of cells is higher than that of suspended electrolyte solutions, and when cells pass through channels, their resistance briefly increases, which increases with cell size. CoulterCounting deviceThe operation is similar to automaticCounting deviceHowever, a blank test must be run before use, and equipment flushing must be performed after use.

pros and cons

KurtCounting deviceFast in speed and capable of accurately counting cells of different sizes, it is commonly used for blood cell counting in hospitals. However, it cannot distinguish between live and dead cells, nor can it accurately count the cells that form clusters. In addition, its maintenance requirements are relatively high.

4 flow cytometer

Flow cytometry is commonly used for more detailed cell analysis, equipped with fluorescence detection technology that can detect labeled intracellular components. Not all flow cytometers can perform cell counting, but flow cytometers with volume measurement capabilities can provide highly accurate cell counting and distinguish cell types through fluorescently labeled antibodies.

pros and cons

Flow cytometers are powerful but expensive, typically ranging from $40000 to $100000 or more, and are therefore rarely used for routine cell counting.

5 Other cell counting methods

Spectral measurements are occasionally used to estimate cell densityDegree. Due to the turbidity of cells, as cell density increases, the amount of light passing through the colorimetric dish decreases. However, due to the fact that the spectrometer does not directly count cells, only measures absorbance, and other variables in the suspension may affect absorbance, this method is not reliable.

Cultivation method is another counting method that is only applicable to cells that form colonies (such as bacteria). After diluting and streaking the cell suspension into a culture dish, count the number of colonies to calculate the cell density of the original suspension. But due to the need to wait for colony growth, cultivation is the slowest method.

Automatic cell counter: More accurate counting brings higher quality science

Diancheng Biotechnology provides you with a variety of high-precision fully automatic cell counters, including bright field cell counters, fluorescent cell counters, automatic bacterial counters, as well as a comprehensive range of consumables and accessories. If you have any related needs, please feel free to contact us!