AAT Bioquest

Cell Preparation Considerations and Troubleshooting

single cell suspension
Basic steps of preparing a single cell suspension from an initial tissue sample. Figure made in Biorender.
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.


Considerations for Cell Preparation

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.

Pipetting, Centrifugation, Wash, and Resuspension

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.

Note: The most effective sample mixing is performed when the pipette is set to approximately 50% of the suspension volume.

centrifugal filtration
Example of centrifugal filtration, using ReadiUse™ 10KD Spin Filter as an example. Spin filters are disposable ultrafiltration devices for the concentration of biological samples such as antibodies. This particular filter has a maximum initial sample volume is ~500 ul. They can be used in either a swing bucket or fixed angle rotors accepting 2.0 ml centrifuge tube at around 10000 g.
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:

  • 1X Phosphate buffered saline (PBS) can be used without calcium and magnesium to prevent cell clumping.
  • 0.1-1% Of BSA or 1-10% fetal bovine serum (FBS) can be added to reduce non-specific binding of antibodies and/or fluorophores to target cells and help maintain cell viability.
  • EDTA (>0.1 mM) and surfactants (i.e. Tween-20) should be limited and/or removed where necessary as these components may interfere with downstream application.
  • Cell clumping may be mitigated by adding DNAse I to reduce aggregation.
  • Cell culture media may also be used in lieu of PBS-based buffers, if there is an issue with maintaining cell viability.


Working with Different Types of Biomaterials

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:
  • A very high level of cell viability (>90%) is recommended as cell loss is likely to occur over time. If sample viability is < 80%, it might be necessary to remove dead cells before freezing.
  • Cell suspension should be frozen, undiluted, in 1 million cells/mL aliquots for ease of resuspension later.
  • Cryoprotectants may be added to the suspension or cryopreservation solutions can be used (sometimes containing up to 10% DMSO) to prevent damage from the freezing process, i.e., ice formation.
  • Frozen cell samples should only be kept in -80 ºC freezers for up to one week and should be moved to liquid nitrogen or <-150 ºC freezers for long-term storage.

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.

Note: Compared to live cell samples, fixed samples can be stored for longer at refrigeration temperatures (up to 1 week) and at lower -80ºC temperatures (up to 6 months) before use in some assays. Although fixed samples may have a longer storage life, they are only compatible with certain assays. For example, they can be used to harvest nuclei but are not recommended for use to generate cell suspensions or adherent populations.

Cleaning up Cell Samples

Fluorescence Activated Cell Sorting (FACS)
Simplified principle of Fluorescence Activated Cell Sorting (FACS), from initial sample loading to marker-facilitated separation. Graphic made with Biorender.
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.

Basis Of DifferentiationFlow CytometryFACS
DefinitionFlow cytometry is an analytical cell biology technique used to identify and study the characteristics of cells in a heterogeneous mixture. It uses differential light scattering properties unique to each cell type in the mixture to determine the number and size of cells and nucleic acid content of the cells.FACS (fluorescence-activated cell sorting) is a specialized type of flow cytometry that facilitates the sorting out cells in a heterogeneous mixture into two or more types. It uses fluorescent-labeled antibodies to specifically identify components of different cell types
Type of techniqueThis is an analytical cell biology technique.This is a specialized type of flow cytometry.
Sampling method usedThe process uses the differential light-scattering properties of cells to collect the necessary data.The process uses highly specific antibodies tagged with fluorescent dyes to distinguish between cell types.
Analysis methodA sensor is used to acquire data.An electromagnet is used to sort the sample.
SequenceFlow cytometry follows FACS.FACS is the first step of analyzing a heterogeneous cell mixture. It is followed by flow cytometry.
FunctionThis technique measures certain cell characteristics such as the number, size, and nucleic acid content of cells.This technique separates cells from a heterogeneous mixture into appropriate subpopulations.

Performing Cell Counting

MycoLight™ 520
Relative viability of E.coli suspensions treated with MycoLight™ 520 were analyzed using the FITC channel of Flow Cytometer. The readings (Count (%)) were obtained from various live/dead E.coli mixtures (A). The live and dead population in each mixture can be easily distinguished by the two distinct peaks. The count of each sample was plotted against the percentage of live bacteria to generate a standard curve (B).
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.



Current best practices in single‐cell RNA‐seq analysis: a tutorial
Cell Preparation for Single Cell Protocols
Principles of protein labeling techniques
Chapter 11: Flow sorting

Original created on February 20, 2024, last updated on February 20, 2024
Tagged under: preparation, troubleshooting