Studying cell populations has become an integral part of performing all different types of biological research. Collecting information on cell behaviors, structures, and quantities can teach us about ourselves and the world around us. Researchers can gather some of this data on a cell population through the use of a technique called flow cytometry.
Flow cytometry is an analysis technique used across many life science applications to identify and measure the characteristics of a cell or particle population. This process allows researchers to rapidly analyze cellular characteristics both qualitatively and quantitatively.
Flow cytometry relies on the use of a device called a flow cytometer. The flow cytometer is made of three separate components:
These three components cooperate to analyze cells during flow cytometry.
To properly harness the flow cytometer there is a specific flow cytometry protocol that must be followed. The flow cytometry steps include the following:
The purification before and/or after cell separation will help to reduce dead cells, cell debris, and other contaminants that could skew results.
Flow cytometry is an analytical technique, meaning after this process the cells can typically be discarded. A motive with FACS is to capture statistics about a particular cell population amongst a sample. Fluorescence activated flow cytometry (FACS), however, is a flow-based cell sorting mechanism that requires an extra step. Essentially, FACS is a cell separation technique that separates individual cells based on their characteristics. When cells are placed into a flow cytometer, they are identified the same way as in flow cytometry but are then sorted into respective “containers.” FACS is the process you would use if you wanted to perform downstream assays on cells after isolating them with a flow cytometer.
As mentioned, a helpful outcome of flow cytometry is provided data on a cell population. A flow cytometer can evaluate thousands of cells per second in a single-cell suspension. Scientists can learn about the volume, size, morphology, count, protein expression health, and lifecycle stage of cells in a biological sample. These physical and chemical characteristics can help scientists learn how to isolate, manipulate, and identify particular cell groups.
When using FACS, the result is multiple enriched cell type populations. These cells could then be studied or experimented on to learn about their behaviors.
While still abiding by flow cytometry principles, there are a multitude of ways to improve the process and optimize results. Here are five solutions you can use to improve flow cytometry results:
Each of these techniques will help you get the most accurate results from your flow cytometer.
Including the right controls involves adding cells to the sample which you can use as a reference to check background interference and how accurate labeling antibodies are. Including negative controls of the same isotype can help you determine background signal in your experiments. Including unstained target cells included with your stained cells will help you control for auto-fluorescence, or cells that are marked as labeled because of the chemicals in the solution. Measuring background interference can help you adjust for it in the results.
The presence of dead cells can skew results if they are labeled with specific fluorescent markers. Scientists use a certain fluorescent dye that can pass damaged membranes to mark dead cells, so they are not misinterpreted as living cells. A sample preparation step to remove dead and dying cells ahead of flow sorting can help to minimize this impact. This will ensure your sorted cells are live and viable if you plan to use them for downstream assays.
The fluorescent signal of a cell is how the flow cytometer recognizes whether or not it has been stained by antibodies. Improving your fluorescent signal is a great way to increase sorting accuracy and improve flow cytometry results. Fluorescent signals can be improved by reducing the background interference from binding antibodies. Determining the minimum amount of antibody can make your fluorescent signals more intense.
Maintaining the intensity of fluorescent signals will also greatly increase sorting accuracy. Preventing phenomenon that negatively affect your signal intensity can be done through the following tactics:
All of these strategies will help prevent the chances of extracellular antigens being internalized when antibodies bind. Essentially, these methods will help reduce random particles from damaging fluorescent signal intensity.
One of the simplest and most effective ways to improve flow cytometry is by improving sample preparation. The more effort you put into preparing samples, the higher your chances of producing reliable results.
There are a number of challenges that present themselves during sample preparation. Flow cytometry is a single-cell process that requires researchers to prevent cell aggregation and maintain cell viability all at once. Failure to meet these objectives while also producing accurate and reproducible results leads to lost time, money, and sample materials.
Samples need to be appropriately prepared prior to processing. Contaminants from blood samples such as RBCs can interfere with cell sorting and increase the overall time required to sort the sample. The fast-flowing liquids have also been known to cause shearing, a form of cell death when the cell membrane is burst from physical stress. Enriching the sample for the cells of interest using an approach like BACS prior to flow sorting can greatly reduce sort times while improving sort efficiencies and downstream results.
Handling these preparation challenges can be done in a variety of ways. Here are some tips that can help you increase the quality of your flow cytometry results:
While some of these strategies require no extra products, others might require you to purchase additional reagents or chemicals.
One of the products you can purchase to help you improve flow cytometry is a BACS microbubble kit. BACS, or buoyancy activated cell sorting is a strategy developed and patented by Akadeum Life Sciences that harnesses the buoyant properties of microbubbles to separate cells. While BACS can be used as a primary cell separation method, it can also be used in conjunction with flow cytometry to improve sample purity, cell viability, and downstream results.
Akadeum’s depletion or negative selection cell isolation products can be used for sample preparation; this sample preparation step can enrich desired cell populations before flow cytometry to make them easier to label. Between high volume, high efficiency RBC depletion kits with a workflow of approximately 10 minutes and negative selection kits that remove any and every unwanted cell without damaging the desired population, BACS can certainly assist you in your flow cytometry sample preparation efforts.
What are some of the benefits of using microbubbles in conjunction with flow cytometry?
Interested in using microbubbles to improve flow cytometry but need more information? Talk to a scientist today to learn more.