Samples of a tissue digest play an important role in various clinical and research applications. When dealing with those samples, viability measurements and accurate cell count before downstream cell-based assays or preparation of samples, are important for obtaining comparable and dependable results in your research. But, the complexity of these samples, which mostly have many cell types of varying sizes and cells, along with cell clusters and matrix/cellular debris, makes it difficult for a cell counter to analyze data. To address this problem, researchers and scientists must improve the accuracy of their cell counters so as to count and identify living cells.
Why Improve Accuracy of Cell Counting?
Most cell sorting and flow cytometry procedures require a specific range of concentration or cell volume for optimal analysis and staining. Incorporating the process of counting cells into flow cytometry workflows is the simplest way of increasing experimental success as well as saving invaluable money and time. This, in turn, enables the following:
Efficiently verify staining
Knowing the number of cells you’ve lost during staining
Determining optimal amount of reagent required for staining
Confirming that you have enough amount of cells for the process
Cell Counting: Manual vs. Automatic
Traditionally, you can manually determine the concentration of cells using a system called a hemocytometer. To use this system, you will have to count cells in the squares of a grid and convert them into cells per milliliter using this equation – number of the counted cells x dilution factor x 10,000 = cells/milliliter. However, this process can take hours. This is why researchers recommend a faster and more efficient method – automated cell counting. An automated cell counter provides accuracy and reproducibility between users within a short period of time. Like most researchers, you will love these two types of automated cell counters: a bright-field imaging counter and a Coulter counter. A bright-field imaging counter has the capability of programming various parameters. Apart from calculating the viability of cells, it also guarantees accuracy/speed, low cost of consumables, and ease of use. On the other hand, a Coulter counter uses electrical currents. If a particle disrupts these currents, it can change impedance. But whichever method you prefer to use, counting of the cells will still need proper preparation for accurate results and reproducible.
Challenges Facing Cell Counting
Characterization of cells depends on optimizing the counting of cells throughout the procedure, from the collection of samples to data presentation and analysis. However, several variables and challenges can interfere with quality of measuring cells before counting even begins. For instance, preparation consistency and sample collection issues might be a problem because of these factors:
Mixing strategies
Vessels
Different collection types
Dyes
Different dilutions
Thawing rates
Tips for Improving Cell Counting Accuracy
Counting of cells underpins many applications, spanning the basics of research through the production and development of a cell therapy. Today, automated cell counting approaches are replacing manual counting of cells, thanks to being more accurate and faster. However, like other approaches, you still need some tips to help you improve the accuracy of cell counting. Some of these tips include the following:
1. Reduce Cell Clumping
Counting cells requires the analysis of small samples of the entire stock solution. This means you must take precautions to ensure your samples are representative of original stock cultures. Although cell counters, such as CellDrop, use an algorithm to identify cells in the clumps, they’re not suitable for non-representative samples. When using a manual cell counter, clumps get more scored than individual cells; thus, increasing the variability of users. Cell debris and extracellular DNA following lysis of cells are major causes of clumps. Cell lysis often results from over/under-digestion, thaw/freeze cycles, overgrowth, and mechanical shearing. By filter your samples and avoiding all those causes, you can minimize cell clumps.
2. Optimize Exposure and Focus Settings
Whether you prefer automatic or manual cell counting approach, it will be best to adjust exposure and focus settings to ensure cells’ visibility. The optimal exposure and focus shows a sharp contrast between the background and cell membranes. For applications that involve fluorescence, the capability of optimizing fluorescence channels may help to ensure reproducible results. The intensities of fluorescence must be set to make sure the cells are bright and their size remains intact.
3. Clean the Surface of the Sample
Ensuring you have a clean surface for your samples is important when counting cells. This is because contamination may result in inaccurate results. Counting cells manually requires cleaning and removing the coverslip of the glass and using a cleanser with 70% ethanol to clean the chambers of your counter, then rinse the surface using water. You will also need new slides for every sample if you prefer using disposable plastic slides.
Because most automated cell counting approaches have the same functions, scientists and researchers might think that they are similar. But the truth is that they have different performances. This is why, apart from these tips, it is also advisable to demonstrate with a couple of models to ensure you get one that precisely counts various samples and improves the cell counting accuracy.
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