Care and technique in cell preparation protocols helps to maintain cell viability and ensures the quality of the sample. It's always important to guarantee that best practices are followed to help minimize the presence of cell aggregates, dead cells, noncellular nucleic acids, and potential biochemical inhibitors that may reverse transcription. Most cell preparation protocols are generally compatible with various cell and sample types, though it may be necessary to optimize steps when dealing with sensitive samples or solid tissues.  

With this in mind, there are a number of general considerations when preparing cell samples. The required input for downstream experiments must be understood, which may include cells and/or nuclei that are, or are not, fixed. In cell suspensions the ideal cell viability is >70%, while in nuclear suspension cell viability should be <5%. In nuclear suspensions, it is also important to make sure that experimental practices protect the integrity of the nuclear membrane.

For all cases, improper or prolonged handling of the cell sample may negatively impact the sample quality. Even samples that sit in suspension buffers for too long may show increased cell loss or the formation of aggregates. The temperature of a reaction may also influence cells, so it is important to prepare cells efficiently and place samples on ice after resuspension to avoid cell shock. This, however, depends on the cell type as some cells, like granulocytes and neutrophils, prefer room temperature conditions.

Proper pipetting techniques, as with most research, is crucial in cell preparation. It is important to pipet suspensions thoroughly, but gently. Single cell suspensions require regular-bore pipette tips, slimmer than wide-bore tips, so that single cells from within pellets or clumps can be easily isolated. Wide-bore pipette tips are useful for other purposes as they help minimize cell stress and the potential for cell shearing during preparation.

 

In centrifugation, optimizing conditions for the specific sample type will help achieve the ideal cell pellet. Centrifugation speeds, durations, and temperatures will impact the cell number, concentration, size, and density of the cell pellet. Even if the pellet does not appear perfect, it is important to remember that excessive centrifugation should be avoided. As stated previously, cell aggregates or impurities like cell debris or fibers can interfere with the quality of samples and provide inaccurate measurements in cell counting. Generally, these can be removed with density driven techniques, filtration methods, or magnetic activated cell sorting (MACS).

When working with crude samples, wash and resuspension steps must also be thought through. An ideal buffer composition is one that prevents, to the largest degree possible, cell clumping, loss, or debris while maintaining cell viability. The buffer(s) used must be in the physiological range between roughly pH 6-8 to limit stress to the cells. Additives, like bovine serum albumin (BSA) can be included into buffers, like PBS, to minimize cell loss, aggregation, and to enhance the health of cells. Importantly, some cell types are more sensitive to medium, for example primary cells, stem cells, or dissociated tumor cells, that may require alternate or optimized wash and resuspension buffers. Below are some steps for a starter wash and resuspension buffer:

It should be obvious by now that mindfully prepared, high quality, starting samples will generate cell suspensions with minimal cell loss. This, too, extends to samples that may undergo freezing and thawing. When freezing a cell sample:

When preparing cells from tissues, it is always important to consult product-specific documents, information in the literature, or general guidelines as varying tissues (soft, solid, necrotic, etc.) must be handled differently. Cell viability in tissue samples is considered adequate at > 70%, and samples may require prior cleaning steps if cell viability is especially low. There are many different protocols available for preparing single or multi cell suspensions from tissues, and one important takeaway from all procedures is that steps must ensure that cells remain as intact as possible.

Cleaning samples by removing dead cells, cellular debris, and other potential contaminants is essential for maintaining a healthy cell population. By reducing unwanted cells and other particulates, the efficiency and accuracy of cell sorting assays will be improved. This, in turn, saves valuable time and resources in the process. Importantly, however, sample cleanup will inevitably result in some cell loss so for smaller or rarer cell populations (<150,000 cells) the number of cleanup steps should be limited.

There are a few common techniques in cleaning up samples, the most notable being through density gradient centrifugation. In this technique, sample components are separated by their size and/or mass density. Commercially, there exist a number of density media and kits that have their own properties for varying experimental needs. Density gradient centrifugation can not only be used to remove cellular debris and aggregates but is also commonly used to fraction peripheral blood mononuclear cells (PBMCs) or isolate plasma from other blood cells.

Cleaning methods can also rely on filtration methods to separate target cells from other components within the samples. Filtration techniques require samples to be pulled through a mesh or sieve-like barrier, where target cells separate from non-target parts of the sample. Common filtration techniques can be used in systems that are passive, centrifugation, or vacuum pump based.

Cell sample clean up can also be performed through bead enrichment techniques, where functionalized beads detect specific moieties of target cells. Bead enrichment is typically performed through magnetic micro-sized beads (e.g., MACS) that work to effectively isolate target cells, proteins, or nucleic acids within a sample. Once the targets are captured, all non-target cells and other persisting cellular debris are washed away from the sample. Bead enrichment uses a reversible magnetic force for bead-cell adherence and separation, limiting the amount of stress put upon the sample during the clean up process. Given the right equipment, this method can be used for an array of sample sizes.

Flow sorting can be combined with each of these cleaning techniques to help ensure a uniform cell suspension. Flow sorting is the general name used to describe cell separation using automated cell sorters, commonly known as FACS, is extremely sensitive, and can help provide a qualitative understanding of the cell sample before and after preparation.

Even after cells are prepared, the job is not quite done. Visualization and quantification of cells in suspension is a critical step in determining if a sample has the appropriate concentration, viability, quality, and cellular dimension for necessary experiments downstream. Most times, this can easily be achieved through use of an automated cell counter, or through the traditional method of manual cell counting through a hemocytometer.

Prior to counting, cells should be pipetted gently to prevent clumping and help provide the most accurate results as cells of different sizes and types will settle and aggregate in solution differently. Debris and extracellular DNA is often present in samples that have undergone lysis and may amplify the occurrence of cell clumping. If not properly removed in previous cell preparation steps, these clumps may cause microfluidic clogs in the cell counting machinery.

To mitigate issues, additional pipetting or filtering techniques may be necessary. Before counting, it is also necessary to input the right specifications into the cell counter which depends on the cells within the sample. For example, a cell counter with only brightfield microscopy optics may be ideal for homogeneous cell lines, while one that is capable of also measuring fluorescence may be the optimal choice for measuring cells of a much smaller size, like PBMCs. At least two independent counts per sample are recommended, and counts are generally considered accurate if they are within at least 25% of each other. The last thing to remember is that once counts are performed, concentration is determined based on the total, not only viable, number of cells.