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Cell counting

In our routine experiments, there are numerous instances where cell density must be calculated in order to determine the inoculation concentration or cell number.
 

Common cell-counting methods include hemocytometer counting, automated cell counters, and flow cytometric absolute counting, among others.

Hemocytometer counting is convenient, rapid, requires few materials, and is low-cost, making it feasible in most laboratory settings. However, the accuracy of the count is highly dependent on manual操作, and since different hemocytometers vary in precision, this can lead to substantial variability in the results.
Cell counters provide relatively stable counts. Various automated cell-counting instruments available on the market are based either on cell imaging or on the Coulter principle, with the primary goal of freeing users from tedious and monotonous manual counting and eliminating operator subjectivity.

Flow-based absolute cell counting is typically performed using one of two approaches: either by integrating data from an independent cell-concentration measurement device within a hematology analyzer with population-level data from flow cytometry (multi-platform analysis), or by spiking the flow cytometry sample with an internal microsphere count standard (single-platform analysis).

 

 

 

 

 

Common methods for assessing cell viability include trypan blue exclusion staining and dual-fluorescence staining; each of these cell counting and viability-assessment techniques has its own advantages and limitations.

The principle of trypan blue staining is that, upon cell injury or death, trypan blue can penetrate the compromised cell membrane and bind to fragmented DNA, thereby staining the cells. In contrast, viable cells are able to prevent the dye from entering. However, trypan blue is a toxic chemical dye; if the staining time is too long, the dye may permeate not only dead cells but also live cells, leading to a reduced cell viability.

AO can penetrate intact cell membranes and intercalate into the nuclei of all cells—both live and dead—resulting in green fluorescence; PI, by contrast, can only cross compromised cell membranes, i.e., those of dead cells, to intercalate into the nuclei of all dead cells and emit red fluorescence. When both dyes are present within the nuclei and used at an appropriate AO-to-PI ratio, energy resonance transfer occurs between them, causing live cells to exhibit green fluorescence in the blue channel and dead cells to display red fluorescence in the green channel, thereby enabling assessment of cell viability. However, fluorescence staining requires specialized cell counters or flow cytometers to obtain quantitative results, imposing stringent equipment requirements.

 

 

 

 

Factors Affecting Cell Count and Viability
The same sample should be counted three times to verify the stability of the sampling.
For cryopreserved cells, proper thawing is essential for maintaining cell viability; improper thawing procedures—such as using insufficient water in a water bath, allowing excessively long thawing times, or removing cryovials before large ice crystals have fully melted—can compromise cell viability.
To verify the accuracy of a pipette, use a deionized water standard (100 µL = 100 mg), aspirate the volume, and then weigh it on a precise analytical balance.
Before aspirating a liquid, first blow and tap the pipette tip to ensure it is fully wetted. For liquids with high viscosity, use the reverse aspiration method: press the aspiration button to the second stop to draw up the sample, then push the button back to the first stop to expel the liquid; discard any remaining liquid.
When using a pipette to take samples, minimize bubble formation to reduce sampling error.
It is recommended to first resuspend the centrifuged cell pellet by gently pipetting in a small volume of buffer, ensuring uniform dispersion of the cells throughout the buffer, before adding a larger volume of buffer for thorough mixing. This step helps prevent cell clumping and ensures complete dissociation of any aggregates.
The absolute cell count is highly dependent on the instrumentation and typically requires calibration to ensure accurate results.
The accuracy of a hemocytometer is determined by its scale precision.
Since AO and PI are nucleic acid dyes that stain only nucleated cells, their cell counts will be slightly lower than those observed under bright-field microscopy.
Trypan blue is cytotoxic, and cell viability is highly dependent on staining duration and trypan blue concentration; improper handling can lead to underestimation of viability.
When performing cell counting, the dilution factor should be accounted for, as the dye will dilute the cell sample.
The cell concentration for counting should be within the effective counting range of the cell counter to avoid significant measurement errors.

 

 

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