Working with cells requires scientists to study large, healthy populations of specific cell types. If there are too many variables in an experiment, it will be impossible to identify which cells may be causing a reaction. One way to ensure the accuracy of downstream results is through the use of additional cell purification methods.
Cell purification is the process of separating unwanted cells from a biological sample to eliminate external influences on downstream results. When attempting to measure or test the behavior of a single cell population, another cell type being present can skew data and provide an inaccurate representation of how the target cells will behave.
When preparing to perform an experiment that requires a single isolated cell population with high cell viability and health, cell purification is a necessary step. This step can be performed before or after the primary method of cell separation to further remove contaminants.
Whether the sample has a variety of unwanted cells leftover from the tissue dissociation phase, or extracellular debris present due to cell death during the cell separation process, cell purification is an easy way to clean up a cluttered sample.
Biological samples often contain numerous cell types at different stages in their development processes. The presence of unwanted or off-target cells could impact downstream analysis and therefore should be removed from the sample if possible.
Some of the most common cells that are known to cause problems in purified cell samples are red blood cells (RBCs) and dead cells.
RBCs will create waste and cause changes in the pH of a solution if left to metabolize, which can alter the behavior of target cells. When isolating cells such as T cells or B cells from a blood sample, a large number of RBCs will likely be present. Removing these RBCs is necessary to run an experiment with accurate, significant results.
Another huge concern when sorting cells is apoptosis, or cell death. When a cell dies, it releases its components into the surrounding mixture. This extracellular debris can group together and cause cell clumping, which makes single-cell suspension or individual sorting very difficult. The more dead cells you have in a sample, the more cells will end up dying as a result of clumping, shearing, and increased sort times. To minimize dead cells, you should consider additional steps to purify your sample and choose a cell separation method that values cell viability.
Depending on the cells you are trying to isolate, some of the other substances you’ll want to purify out of a sample may include the following:
Once you know what you would like to remove from your sample, you can more accurately purify your sample before or after cell separation.
Purifying a cell culture can be done in a multitude of ways. When it comes to cell purification, affinity molecules such as antibodies are currently the gold standard for isolating heterogenous mixtures with high purity. Antibody binding-based methods are techniques that mark target cells with antigen-specific antibodies to make them more noticeable or accessible during the rest of the process.
Two of the most popular antibody binding-based methods are magnetic bead-based cell sorting and fluorescence-activated cell sorting.
Magnetic bead-based cell sorting uses a magnetic field in conjunction with antibody-coated microbeads that attach to target cells. This method can be used for isolating rare samples and high throughput purification for highly concentrated cell populations. MACS requires additional equipment in the form of a magnet or magnetic columns to properly sort a sample. When the antibodies bind to target cells, they are subjected to a magnetic field that suspends them. After removing the labeled cells, the magnetic field is turned off so the enriched substance may be collected.
While MACS has a high throughput and is relatively inexpensive in comparison to some other methods, it’s not always the best option. MACS is dependent on the availability of antibodies to isolate specific cells. The harsh magnetic field can also damage rare or fragile cell populations, causing them to rupture or lyse. The cellular debris released from these dead cells can contaminate the purity of the sample and decrease its overall viability. After MACS the target cells are also still attached to the magnetic microbeads and must be separated before any other downstream processes can occur. Due to some of these factors, magnetic bead-based separation can limit the throughput and overall effectiveness of sample cleanup prior to downstream processing.
Magnetic bead-based sorting is often paired with another method to further purify a high throughput sample. Following up a sorted sample with a second, more precise method can salvage decent results if enough of the desired cell population remains. BACS can be applied to an isolated sample to reduce cell debris, other contaminants, and ultimately increase the purity of a sample.
Fluorescence activated cell sorting uses a modified flow cytometer to isolate cells and purify samples based on both physical and chemical characteristics. Prior to isolation, the cells in a mixture are labeled with fluorescent antibodies that bind to the surfaces of cells of interest. The cells are then quickly streamed through a nozzle one by one where a laser measures their light scatter and fluorescence-associated features. If a cell meets predetermined criteria for a target population, the flow cytometer will automatically sort it into its respective group.
FACS is particularly useful for sorting multiple targeted cell populations simultaneously. Even populations with low frequencies are typically sorted with high purity in a sample. However, issues with FACS arise when trying to isolate a large number of cells from a low frequency cell population. This method can become complicated if the concentration of cells is too high. The flow cytometer may make a mistake and sort two unique cells into the same group on accident. If the concentration of target cells is too low the process will take a very long time and risk cell viability, causing cellular debris to taint results.
When purifying a cell culture with FACS, a successful throughput depends heavily on the state of the input sample. The presence of any cell debris or unwanted contaminants that may affect the sorting process can be detrimental to downstream results and assays. To assure that a sample has high cell viability and sample purity before being sorted in the flow cytometer, use an additional method like BACS to clean up your mixture.
BACS adds almost no time to the process with the negative selection workflow from setup to purified sample taking 30-45 minutes. Using microbubbles can end up saving you time by increasing the speed of your fluorescent sorting process and preventing mistakes that would require a second experiment. By using BACS to prepare a heterogenous mixture for FACS, you can set yourself up with a clean and healthy sample that can be sorted quickly and accurately.
Using the negative selection strategy from these microbubbles to help isolate immune cells ensures that the final sample is pure and will behave accordingly to the specific cell type.
In the fields of medicine and immunology research, the study of T cells is how scientists discover and test new diseases and treatments. There are a variety of different T cells that each play a significant role in creating the human immune response and understanding how they work is an integral part of individualized medicine.
Akadeum currently offers various T Cell Isolation Kits, four for murine T cells and two for human T cells.
Among the T Cell Isolation Kits offered by Akadeum are the Mouse Naïve T Cell Isolation Kit, Mouse Naïve CD4+ T Cell Isolation Kit, Human T Cell Isolation Kit, Mouse T Cell Isolation Kit, Human CD4+ T Cell Isolation Kit, and the Mouse CD4+ T Cell Isolation Kit.
B cells are another type of immune cell that also play a huge role in the human immune system. More specifically, B cells deal with antibody production and building up long term immunities.
Akadeum offers a Mouse B Cell Isolation Kit that offers a fast way to isolate mouse splenocytes for downstream applications.
Microbubbles can be used to further purify samples that have been separated using other isolation methods, or they can be used as the primary method. Akadeum has developed innovative BACS microbubble kits that combine a high purity, sample viability, and throughput to delete the need for additional purification steps. BACS kits have no harsh magnetic fields or fast-flowing liquids that jeopardize cell health and physiology.
For more information on why Akadeum’s microbubble kits are better for your cell separation efforts than other antibody binding-based separation methods, check out our page on Evaluating Cell Separation Techniques.
If you’re still curious about what microbubbles are and how they work, download our Ultimate Guide to Microbubble Technology and Cell Separation.