When isolating cells with Dynabeads magnetic beads, what is more important: bead-to-target cell ratio or the concentration of beads in the bead/cell mixture?
Both bead-to-target cell ratio and the concentration of beads in the bead/cell mixture are important and should be considered. For example, when using the Dynabeads magnetic beads M-450 CD4 positive isolation or depletion kit, a 4:1 bead-to-target cell ratio should be maintained. To capture 95% of target cells for molecular applications, the bead concentration must always be 1 x 10e7 beads per milliliter of sample. To deplete 99% CD4 cells from the starting sample, the bead concentration must always be 2 x 10e7 beads per milliliter of sample. Please consult the package insert for recommended bead concentrations of each product.
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Will Dynabeads magnetic beads be internalized if cultured with the beads on?
Whether cells will internalize the Dynabeads magnetic beads during culture will depend on the cell type. Due to the bead size (usually 4.5µm in diameter) Dynabeads magnetic beads will not be internalized into the endocytic pathway e.g., via clathrin coated pits. The clathrin coated pits are typically not more than 500 nm in size, which is far too small for endocytosis of the beads. However, if cells with phagocytic activities (e.g., monocytes/macrophages) are present, the Dynabeads magnetic beads will be phagocytosed into the phagolysosomes by these specialized cells. So it would really depend on the cell type.
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Is there a method to remove the Dynabeads magnetic beads from isolated cells if the bead releasing reagent is not available?
We offer several Dynabeads magnetic beads that can be used for either positive isolation (keep the target cells) or for depletion (remove the target cell from a sample) that does not include any release mechanism:
- Dynabeads magnetic beads for depletion: Using Dynabeads magnetic beads for depletion is a very fast, efficient and easy method. Use pre-coated Dynabeads magnetic beads or coat your own target antibody onto our secondary coated beads, add to any sample (e.g., whole blood, PBMC, buffy coat, tissue digests), incubate for 20 minutes with mixing, apply to a magnet for 2 minutes, and you have your cells depleted.
- Dynabeads magnetic beads for positive isolation for molecular downstream assays: Positive isolation of target cells without bead release can be used when the aim is downstream molecular studies such as DNA, RNA, or protein analysis. In these applications, the isolated cells can be lysed while the beads are attached to the cells, and the beads can be removed after cell lysis. If the bead presence is not a problem, you can also culture the cells with the beads on. In most cases the surface antigen will be internalized after 2-3 days, and then the beads will fall off since the beads are too big to be internalized by the endocytosis pathway.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
Will Dynabeads magnetic beads be internalized by the target cells?
In general, the size of the Dynabeads magnetic beads is so large that they will not be internalized. The clathrin-coated pits are typically not more than 500 nm in size, which will be too small for Dynabeads magnetic beads to be internalized by endocytosis. However, if the target cells have phagocytic activities such as monocytes/macrophages, the Dynabeads magnetic beads could be internalized by phagocytosis.
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How do Dynabeads CELLection cell isolation kits work?
The CELLection kits contain Dynabeads magnetic beads coated with a DNA linker (either via streptavidin or via an antibody for cell isolation) and a DNase enzyme for bead release. After the beads capture the target cells (either directly or indirectly), the cells are released by DNAse treatment to cleave the linker. As a result, the target cells are released from the Dynabeads magnetic beads, but still linked with capture antibody.
We offer three Dynabeads CELLection cell isolation kits:
- CELLection Epithelial Enrich Kit (Cat. No. 16203) contains Dynabeads magnetic beads coupled with anti-EpCAM monoclonal antibody
- CELLection Biotin Binder Kit (Cat. No. 11533D) is used for positive isolation of target cells with your own biotinylated antibody
- CELLection Pan Mouse IgG Kit (Cat. No. 11531D) is used for isolation of target cells with your own mouse IgGs
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
