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CD4 Helper T Cells: What Are CD4 T Cells? CD4 T Cell Functions and Isolation Methods

Updated on Aug 21, 2023

What are CD4 Cells?

The human immune system is broken down into two major categories: the innate immune system and the adaptive immune system. The innate immune system is comprised of different physical, chemical, and cellular responses to pathogens that jump into action, offering general and non-specific first line defense when the immune system is called to act in an effort to contain the spread of pathogens. The adaptive immune system, on the other hand, is acquired immunity that has been developed to recognize, deal with, and develop a response to specific antigens.

A multitude of cells are responsible for carrying out the immune processes that allow our bodies to fight off infection and disease. T lymphocytes are one of the primary cell types that help to mediate cellular immunity for the adaptive immune system. A majority of T lymphocytes are CD4+ and CD8+ T cells.

CD4+ cells are some of the first to recognize harmful substances and notify the body of their presence. These white blood cells are also called helper T cells because they help other cells carry out their specific roles. These cells work with different types of T cells to protect the body.

CD4 Cells Function in the Immune System

CD4+ T cells are not directly responsible for neutralizing infections or destroying harmful pathogens in the body. Instead, these helper T cells secrete chemicals to trigger the body’s immune response. Depending on the specific cytokines the CD4+ cell releases, it will activate different immune cells to come deal with the threat. Although these cells don’t do the actual combating, their ability to identify pathogens paves the way for other cells to handle the infection. CD4+ cells also play a critical role in suppressing the immune reaction after the response is over.

Research Purposes for CD4 Helper T Cells

On top of being a necessary component to the bodily immune response, CD4+ T Cells are also valuable outside of the body for research and diagnostics. By studying isolated CD4+ lymphocytes, scientists can gather information about the patient they came from and study the immune response.

A simple blood test called a CD4+ count is used to approximate the number of healthy, functioning helper T cells in one cubic millimeter of blood. In a healthy adult, the average count is usually between 500 and 1500 cells per cubic milliliter. Significant variations in this number can be indicative of issues in the immune system, such as the presence of human immunodeficiency virus (HIV).

Isolated CD4+ cells are also used in laboratory research to test how the immune system reacts to certain diseases or treatments. Simulating exposure to harmful pathogens with a CD4+ culture can provide insight into potential therapeutic approaches that could be effective in fighting particular infections. These experiments can guide the way to more specialized treatment that will positively influence patient outcomes in the future.

Isolating CD4 Lymphocytes with Traditional Cell Separation Methods

The process of isolating T cells is an important step in many types of cellular research. To properly conduct experiments, external factors such as unwanted cell populations must be eliminated. Otherwise, these additional variables could affect the outcome and skew downstream results.

There are multiple options for cell separation techniques and approaches. Depending on the specifics of a given experiment, it may be more beneficial to use one method or another. Some of the more commonly used methods include:

  • Magnetic activated cell sorting (MACS)
  • Fluorescence activated cell sorting (FACS)
  • Density-gradient centrifugation
  • Buoyancy activated cell sorting (BACS)

Each of these have their own advantages and, in some cases, disadvantages in enriching samples for CD4+ T cells.


Magnetic activated cell sorting (MACS) uses a magnetic field to suspend antibody-marked particles in a fluid sample as other cells pass through. There are two common approaches to a MACS workflow:

  • Column-based – A magnetically-labeled sample is passed between magnetic columns that create a field to capture cells bound to magnetic beads.
  • Column-free – A tube containing a magnetically-labeled sample is placed in a magnetic field, causing the magnetic bead-bound cells to be held in place at the side of the container while the remainder of the sample is poured out.

Magnetic processing exposes cells to magnetic gradients, shear forces, and rare earth magnets, which can have detrimental effects on delicate cells and can impact the health and viability of the final cell population. The necessity of magnets, columns, and other consumables means that a MACS workflow is equipment-intensive and requires the lab to have these components easily accessible to perform the cell separation.


Fluorescence activated cell sorting (FACS) relies on fluorescent markers and a flow cytometer to sort cells according to their physical characteristics. By measuring the light scatter caused by a laser, a modified flow cytometer can separate cells into their respective groups one by one.

This process is beneficial when sorting a heterogenous mixture into multiple individual cell populations, but is not practical when working in smaller laboratories or with smaller samples. The equipment and training are expensive and the process takes a very long time compared to other methods. The fast-moving liquids also cause some cells to shear, or rupture.

To optimize FACS results, a sample preparation step using an enrichment method like BACS can be performed to enrich the sample for the cells of interest. By removing off-target cellular contamination prior to sorting, overall sort times can be greatly reduced leading to better results and a more robust population of cells for downstream use.

Density-Gradient Centrifugation

Density-gradient centrifugation uses a device called a centrifuge to spin samples, facilitating separation of the components within the sample based on physical properties such as size and density. An extra reagent, or substance used in a chemical process, is added to act as a buffer between target cells and unwanted cells.

Similar to FACS, this method requires expensive equipment and can damage cells. While it’s very useful for sorting large volumes of cells, depending on the size of the centrifuge, the harsh processing can make density-gradient centrifugation ineffective as a method of cell isolation when working with delicate cell types, especially when cell health and viability is critical to the success of downstream processing.

BACS using Akadeum’s Microbubbles

Akadeum Life Sciences has developed an innovative technology that harnesses the buoyant properties of small, solid microbubbles to bind to and float target analytes to the top of a sample container. This process, called buoyancy activated cell sorting, or BACS, offers a fast and easy workflow that is exceptionally gentle on delicate cells. Microbubbles can be functionalized in a variety of ways to allow for capture of diverse analytes.

Using microbubbles for cell separation eliminates the need for additional equipment. The functionalized microbubbles are simply mixed into the sample where the bind to their target analyte(s) before gently floating to the surface of the sample container, bringing with them their bound targets for removal. The rest of the sample remains behind, untouched and ready for downstream use.

Because BACS harnesses the power of gravity (the bubbles float) for separation, enrichment is performed directly in the sample container and does not require that delicate cells of interest be exposed to harsh processing, shear forces, and rare earth magnets. The microbubble workflow is exceptionally gentle on cells, maintaining cell health and physiology during enrichment and resulting in an incredibly pure population of rare and delicate cells for downstream applications.

BACS and CD4 T Cells

Akadeum’s Human CD4+ T Cell Isolation Kit uses streptavidin-coated microbubbles in combination with an optimized biotinylated antibody cocktail to selectively label, capture, and remove unwanted cell populations. The unwanted cells are first labeled with the provided antibodies, and then bound to the streptavidin-coated microbubbles using a fast and gentle mixing step. The unwanted cells, bound to the microbubbles, are floated to the top of the sample container for removal while the highly-enriched population of untouched CD4+ T cells remains behind for downstream processing. This fast, easy, and exceptionally gentle protocol significantly reduces overall sort times while maintaining the health and physiology of CD4+ T cells.

To see an example of how Akadeum’s Human CD4+ T Cell Isolation Kit can help to achieve a 15-fold reduction in enrichment time without sacrificing purity, you can download our App Note: Combining Microbubbles and Cell Sorting for Quick & Gentle Isolation of Unique Cell Populations. Download this app note to understand how to successfully isolate cells of low abundance, while saving time and maintaining high viability, through a combination of Akadeum’s microbubble based CD4+ T cell enrichment and subsequent Treg isolation using NanoCellect’s WOLF® Cell Sorter.

If your Human CD4+ T cell enrichment workflows could benefit from the fast, easy, and gentle microbubble workflow, we’d love to hear from you! Contact us and a member of our scientific team will be in touch to learn more about your application and whether microbubbles could help you overcome long-standing headaches in sample preparation. Our team of experts is here to help you develop a workflow that optimizes results, helping you isolate an ultra-pure population of happy, healthy CD4+ T cells for your application.

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