Using Microbubbles to Improve Flow Cytometry: Protocol for Using BACS with Flow Cytometry

Using Microbubbles to Improve Flow Cytometry: Protocol for Using BACS with Flow Cytometry

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.

What is 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 Procedure

Flow cytometry relies on the use of a device called a flow cytometer. The flow cytometer is made of three separate components:

  • The Fluidics System –This portion of the device is responsible for transporting a single-file stream of cells from the sample cell into the flow cytometer.
  • The Optics System – Made up of lenses, laser, filers, and other pieces that generate a photocurrent with light. The device uses this system to detect the characteristics of individual cells as they pass through.
  • The Electronics System – The data gathered from the optics system is then digitized and processed into data representative of the overall sample.

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:

  1. Purify the cell sample for optimal throughput and accurate reporting.
  2. Add fluorescent markers to the solution that will bind to target cells and help them be recognized by the optics system.
  3. Run the cell sample through the flow cytometer in a single-cell stream.
  4. Record the information from the electronics system to gather information on the cell populations involved.
  5. Depending on whether the final step was an analysis, or an additional modification was used to perform fluorescence activated cell sorting (FACS), discard or collect the cells.
  6. Further purify collected samples if necessary.

The purification before and/or after cell separation will help to reduce dead cells, cell debris, and other contaminants that could skew results.

FACS

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.

Flow Cytometry Result

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.

Improving Flow Cytometry

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:

  • Choose the right flow cytometry controls
  • Obtain meaningful data from viable cells by staining dead ones
  • Reduce interference from your fluorescent signal
  • Maintain the fluorescent signal
  • Use the correct methods for sample preparation

Each of these techniques will help you get the most accurate results from your flow cytometer.

Cytometry Controls and Dead Cells

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.

Fluorescent Signals

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:

  • Work with antibody solutions that are aggregate-free
  • Include chemicals such as sodium azide in the staining buffer to reduce cellular metabolic activity during staining
  • Keep your samples refrigerated or ice-cold

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.

Flow Cytometry Sample Preparation

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.

Sample Preparation Challenges

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.

Sample Preparation Solutions

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:

  • Sample Storage – For short-term storage of samples, use a reagent that allows them to be stored at 4 degrees Celsius rather than freezing to avoid possible cell death.
  • Cell Detachment – When removing cells from a culture dish, don’t scrape them, use other enzymatic methods. Scraping the cells could damage the membranes.
  • RBC Removal – Red blood cells (RBCs) can overwhelm flow cytometers and absorb antibodies. For these reasons, they need to be removed from a sample ahead of time for optimal results.
  • Filtration – Using a small cell strainer to filter out cell clumps and aggregates can increase sorting accuracy of the flow cytometer.
  • Mixing – Thoroughly mixing a sample ensures that the labeling antibodies have interacted with as many target cells as possible. If unmixed, certain target cells may go unmarked or unnoticed.
  • Gentle Washing – When performing routine washes, do not centrifuge for too long and avoid harsh conditions that could harm cells.
  • Cell Counting – Using an automated cell counter as opposed to a hemocytometer can increase accuracy. The hemocytometer may allow you to see cells, but it also introduces human error and approximation into the experiment.

While some of these strategies require no extra products, others might require you to purchase additional reagents or chemicals.

Flow Cytometry Techniques

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?

  • The use of microbubbles for sample preparation can increases throughput and processing speed
  • Microbubbles help to maintain the health, viability, physiology of cells by being exceptionally gentle on delicate samples
  • Using negative selection, microbubbles offer high sensitivity, improved scalability, and a greater recovery rate of rare cells
  • The microbubble workflow can be performed quickly and directly in the sample container; a few extra minutes at the beginning can save a large amount of time and resources down the road

Interested in using microbubbles to improve flow cytometry but need more information? Talk to a scientist today to learn more.

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