Bead Engulfment Assay

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Phagocytosis is a process in which target cells or particles are engulfed and taken up by other cells, typically professional phagocytes; this process is crucial in many physiological processes and disease states. Bead engulfment assay is commonly carried out in virus or tumor studies, to assess the phagocytic potential of activated effector cells. As an industry-leading global CRO company focused on CAR-MA therapy, Creative Biolabs tests phagocytosis by bead engulfment assay to assess the direct antitumor activity of CAR-MA.

Introduction of Phagocytosis

Phagocytosis is the mechanism by which relatively large (>0.5 μm) particles, such as bacteria, dead cells, or beads, are internalized. Human macrophages are capable of phagocytosing targets that vary over about a 30-fold range in size and almost 10⁹-fold range in rigidity. Phagocytosis comprises a series of events, starting with the binding and recognition of particles by cell surface receptors, followed by the formation of actin-rich membrane extensions around the particle. Fusion of the membrane extensions results in phagosome formation, which precedes phagosome maturation into a phagolysosome. As phagocytes, macrophages identify phagocytic targets using receptors displayed on their plasma membrane. Once a phagocytic receptor binds its ligand, a coordinated intracellular signaling cascade can lead to phagocytosis or the decision to ignore the object (e.g., SIRPα–CD47 checkpoint).

Fig.1 Phagocytosis of a target particle. (Richards, 2014)

Bead Engulfment Assay

To assess the engulfment of macrophages expressing the appropriate CAR, we use silica beads coated with a supported lipid bilayer binding the target antigen. CAR-MA can recognize and attack beads covered in proteins found on cancer cells. The beads and CAR-MA are visualized with fluorescent labeling the supported lipid bilayer or the cell membrane respectively. The detection of the phagocytosed beads can be carried out using flow cytometry, fluorescence technology or electronic microscopy (EM) in the bead-based engulfment assay. Engulfment is quantified as the mean beads eaten per cell. As expected, CAR-MA can trigger the engulfment of silica beads, indicating CAR expression in CAR-MA. To further define the capabilities of the CAR-MA, we assess the capacity of CAR-MA to engulf variably sized targets. We provide beads ranging from 2.5 µm to 20 µm in diameter, with higher specificity above background engulfment.

As a collaborative CRO partner, Creative Biolabs delivers integrated bioanalytical solutions for your toughest needs. We run not only various phagocytosis assays. Cargo for phagocytosis includes bacteria, beads, or tumor cells. We also but also offer high-quality Bites Assay and Eating assay. We strive to provide excellent data in a timely, collaborative manner by outstanding scientists. If you have any questions or demands, please don't hesitate to contact us.

Reference

Richards, D.M.; Endres, R.G. The mechanism of phagocytosis: two stages of engulfment. Biophys J. 2014, 107(7): 1542-1553.

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