Choosing the correct cell separation method for your laboratory can make all the difference in ensuring accurate results. We’ve reviewed some of the available cell separation kits to make your decision easier.
There are a variety of mouse T cell separation kits available that will allow you to extract an isolated sample of T lymphocytes from murine tissue. Performing T cell isolation from mouse spleens supplies a high concentration of murine white blood cells (WBCs) which function similarly to human WBCs.
Scientists use murine T cells as a widely available substitute for human cells that can be tested to provide insight into how the human immune system may respond when exposed to certain pathogens or substances. Due to the popularity of mouse T cell separation kits, a multitude of biotechnology companies sell products that harness different strategies to carry out the process.
The methods for isolating mouse T cells are the same that are typically used for human cells. The three techniques covered in this article are fluorescent sorting, magnetic cell separation, and microbubble isolation.
Fluorescence activated cell sorting (FACS) involves the use of a flow cytometer device that sorts cells based on their physical characteristics by shining a light through them. After analysis, the cells are split into their respective populations based on traits, grouping and isolating multiple unique samples at once.
Magnetic bead-based cell sorting is a form of isolation that captures labeled cells in a magnetic field while letting others pass through. The cells are first labeled with affinity molecule coated magnetic beads which bind to target receptors. Then, regardless of whether the cell separation kits are column-based or column-free, the sample is subjected to a magnetic field that suspends target cells. Cell separation using magnetic beads is the most common technique for murine T cell isolation due to the wide variety of cells it can target.
Column-based magnetic cell sorting requires the researcher to buy complex equipment for the process to take place. The magnetic columns are positioned on either side of the solution to create a magnetized wall.
One of the alternatives to column-based MACS is column-free MACS, which uses a to separate the cells within a tube. The magnet pulls labeled target cells to the sides of the container, and the unwanted cells are poured out. Then, the field is turned off and the tube has only the desired substance left at the bottom.
While this method is easier, the magnetic field created in column-free MACS is weaker, which in turn requires target cells to be labeled more heavily. This massive labeling can activate or alter certain cells, skewing certain downstream results.
Another alternative to MACS and FACS is BACS, or buoyancy activated cell sorting. BACS is a cell separation technology developed by Akadeum Life Sciences that harnesses the properties of small, polymer-shelled glass bubbles to float target cells to the top of a sample. Similar to MACS, these microbubbles use affinity molecules to attach to biological analytes. Once attached, the microbubbles gently lift cells upwards, leaving behind the enriched sample untouched at the bottom of the container.
Microbubble kits are less expensive, faster, and better for maintaining cell physiology than other separation methods.
When comparing cell separation kits, it’s important to use the correct metrics. Different kits are better for different situations and choosing the right one is a matter of research and consideration.
The purity of a sample refers to the concentration of the desired substance after the isolation process. This value is typically expressed as a percentage. For example, if you are attempting to isolate spleen T cells through mouse lymphocyte isolation, your final purity will be judged on the amount of murine T cells present in the sample divided by the total number of cells. If you isolate 100 cells and 80 of them are murine T cells, while the other 20% are a mix of red blood cells (RBCs), plasma, or other contaminants, the sample purity will be 80%.
Retaining a good sample purity is necessary when performing downstream analysis or experimentation on isolated samples because it ensures that other cells are not confounding the target population’s behavior.
Two other qualities that are directly related to one another are cell yield and recovery. Cell recovery refers to the proportion of desired cells that were successfully extracted from an isolated sample in comparison to the total number of desired cells that were available in the original cell population. Cell yield refers to the total number of desired cells separated from the isolated sample. The higher the cell yield, the higher the recovery, and vice versa.
These traits pertain to the actual number of cells you will have to manipulate after cell separation. A high cell yield and recovery are essential when studying cell behavior.
While most of these traits speak for themselves, there are also hidden benefits to each of them. For example, while the speed of a separation process is convenient, it also preserves cell health. The longer cells are subject to the environmental conditions of a magnetic field, or flowing liquids, the higher chance they will not survive. Workflow and ease of use can save money for training personnel, save time for other processes, and save a headache for everyone involved.
Speed, workflow, and ease of use are not worth sacrificing purity, recovery, and yield. However, certain cell separation methods offer these benefits as well as a good throughput.
At the heart of all scientific research is funding. Unfortunately, there is never quite enough to go around, which means cost is a factor. The price of a cell separation kit is a serious consideration when the monetary resources for an experiment are limited.
Trying to balance all of these qualities and find a decent price can be extremely difficult. The ideal cell separation method should provide all the above and still be practical for small labs or independent researchers.
The context of an experiment is the single most important thing to consider when choosing a cell separation kit. Different methods are more effective in different scenarios, and sometimes it’s necessary to settle for low performance in one category if there are no other options.
Taking all these metrics into account, here are some products we’ve decided are the best fit for their respective cases.
If your laboratory is looking to isolate splenocytces T cells, naïve T cells, CD4+ T cells, or naïve CD4+ T cells from mice the best overall choice is to go with Akadeum’s BACS microbubble kits.
Each mouse cell isolation kit can process 10 samples at 100 million cells per sample.
With no additional equipment required beyond the cell separation kits, these kits are the least expensive. They also have a 30-minute workflow from start to finish and can be done directly in the sample container.
Beyond the practicality of Akadeum’s cell separation kits, they also have a high yield with up to 96% purity. The accuracy and efficiency allows all unwanted cells to be removed by negative selection, leaving the enriched sample untouched at the bottom of the container. Leaving these cells untouched preserves their health and physiology for downstream applications.
STEMCELL Technologies has an extremely diverse set of cell separation kits including magnetic-based streptavidin beads, STEMCELL CD4+ isolation kits, and many other magnetic cell separation products. Magnetic beads have been around longer than BACS and are currently capable of targeting a larger variety of cells. The best kits for sorting less common mouse lymphocytes are the column-free EasySep magnet kits. STEMCELL’s EasySep products are a quick and efficient way to isolate murine T cells.
The actual sorting process can take between 10 and 30 minutes depending on the target cells, and purity typically falls between 91-98%.
While able to target a diverse range, EasySep uses a harsh magnetic field that can damage fragile cells. Cell yield and recovery are harmed by cells lysing, or rupturing, due to the magnets. Beyond this concern, the total price for this product is much higher than Akadeum’s BACS kits.
Although there are no cell separation kits that use FACS as a sorting method, it has been included as the best use case when sorting multiple cell populations at once. Without processing a sample multiple times, neither BACS nor MACS can isolate multiple cell populations in one sortation.
FACS is much more expensive than other methods and requires complex machinery that can cost anywhere between $80,000-500,000. The sorting process takes much longer, and personnel must be specifically trained to handle this machinery.
FACS relies almost solely on the flow cytometer to device for cell separation. While additional products and fluorescent markers need to be bought for cell labeling purposes, they are not considered cell separation kits because they only aid in the process.
Cell Separation Kits Vs Cell Separation Machines: Automated or Manual Cell Separation
Along with more and more manual cell separation kits, another form of separation is also becoming more common. Many biotechnology companies are developing cell sorters that automate the isolation process. These machines function more like a flow cytometer, with reagents being added at the beginning of the process then the device doing the rest of the work.
Depending on how often your laboratory performs cell separation, it could be worthwhile to invest in an automated cell sorter. Especially if you are routinely reliant on more complicated practices such as MACS to sort rare cell populations.
The downside of automated sorting is how expensive it is not only to purchase the machine, but to repetitively purchase the reagents and buffers required for separation.
Below are some of the honorable mentions in cell separation products that still offer specific benefits. Depending on your experiment, one of these products might be more useful than the ones mentioned above.
Miltenyi Biotech is one of the larger companies still using column-based MACS as their primary method of cell separation.
The AutoMACS Pro Separator is an automated cell sorter that can sort the same mouse T cells as Miltenyi microbeads. This device will increase the speed and purity of processing but is expensive, plus the cost of reagents and buffers must be factored in.
ThermoFisher Scientific is a reliable company involved in a wide variety of fields beyond cell separation and isolated cell research. Their Dynabeads magnetic beads require no columns and provide a high yield, purity, and viability for the recovered cells. Similar to other MACS products though, their recovery is lower than other methods due to the harsh magnetic field.
BioLegend is another biotechnology company that sells primarily column-free magnetic-bead based products. The purity of these beads is around 96%, placing it towards the middle of the pack.
BioLegend offers a slightly cheaper alternative compared to other magnetic cell sorting products, however they are not as heavily reviewed and trusted.
When performing mouse T cell isolation with a single target cell population, the two primary methods are BACS and MACS.MACS kits require additional products and hidden costs that make it significantly more expensive than BACS.
Beyond the financial price, when using a magnetic field to suspend fragile cells, you must pay the price in cell yield and recovery. While the purity of MACS is still fairly high, the number of cells that will survive is lower than BACS. When performing downstream tests or experiments, having the highest isolated population possible is how you set yourself up for success.
BACS is cheaper than any alternative, but that doesn’t mean it’s always the correct option. Due to its youth as a cell separation method, BACS is not capable of targeting as many specific cells as magnetic bead-based cell separation. When dealing with certain cells, microbubbles may be completely out of the question.
If this is the case, the amount of money your laboratory has, whether or not you have space for a machine, and the demand for cell separation will all be factors in choosing the right cell separation kit or device.
For cells that BACS can target, it is the most cost-effective and efficient method. When performing mouse T cell and CD4+ isolation with the intention of downstream analysis or applications, Akadeum’s microbubbles provide the most practical solution. Other T cell separation kits cannot offer the same performance at such a reasonable price.
If you’re interested in Akadeum’s technology or would like to partner with our company, To learn more about how BACS compares to other cell separation methods, check out our page on Evaluating Cell Separation Techniques or download our Ultimate Guide to Microbubbles Technology and Cell Separation.