Can you provide an overview of epithelial cell enrichment using the CELLection Epithelial Enrich kit?
CELLection Epithelial Enrich reagent is bound to the beads via a cleavable DNA linker. This provides you with the option to release the isolated cells from the beads using the supplied release buffer, which returns cells with no beads attached for further cell culture or functional assays. CELLection Epithelial Enrich reagent is recommended for processing a total of 2 x 10E9 cells. The CELLection Epithelial Enrich beads are coated with a mouse anti-BerEP4 monoclonal antibody to the human epithelial cell adhesion molecule (EpCAM).
By making a single-cell suspension of the tissue, it should be possible to isolate the cells using CELLection Epithelial Enrich reagent (for cellular applications) or Dynabeads Epithelial Enrich reagent (for molecular applications). The difference between the CELLection Epithelial Enrich product and the Dynabeads Epithelial Enrich product is that the primary antibody on the CELLection product is coupled to the beads via a DNA linker, providing this bead with a release mechanism via the DNase containing release buffer supplied with the kit. This product is intended for isolation of cells that need to be bead free for downstream studies, while the Dynabeads Epithelial Enrich product is intended for molecular applications (e.g. DNA or mRNA isolation). However, since the antibody coated onto these beads recognizes EpCAM, this epitope needs to be expressed on the cells.
The CELLection Epithelial Enrich product is intended for enrichment of tumor cells rather than isolation of pure cells. This is because the number of target cells can often be as low as 1 tumor cell in one million cells, making it very difficult to get rid of all the contaminating cells even after rigorous washing. This is why we also recommend that molecular analysis or visual morphological verification is necessary to verify that the isolated cells are indeed tumor cells.
How can I capture cells successfully using CELLection Dynabeads magnetic beads?
To ensure the most efficient cell capture:
Always titrate the antibody concentration used for coating the CELLection Dynabeads magnetic beads and test the direct and the indirect technique. We suggest using 1 x 10e7 beads per mL sample (25 µL) for efficient positive isolation. Incubate at 2 - 8 degrees C with tilting and rotation. When using whole blood samples, wash whole blood as described before use.
To ensure the most efficient cell release:
Never vortex DNase when enzyme is in solution, as this may destroy enzymatic activity. For cell release, use fresh RPMI plus 1% FBS prewarmed to 37 degrees C to ensure the DNase is active. The pH of the RPMI should be 7.0 to 7.5. Higher pH will inhibit DNase activity. Optimal DNase activity requires divalent ions (Mg2+, Ca2+, Mn2+). Generally, no increased release is observed when additional divalent ions are added to the DNase Releasing Buffer, but for this option, use one part of 10x Tris to 9 parts RPMI. (10x Tris = 400 mM Tris-HCl, 100 mM MgSO4, 10 mM CaCl2, 10% fetal calf serum). After cells are incubated with DNase Releasing Buffer, it is essential that the bead-cell complexes are vigorously pipetted before magnetic separation to mechanically disrupt the DNA linker. Failure to pipette the cells will affect cell yield.
Can I store the antibody/ligand-coated Dynabeads magnetic beads?
Dynabeads magnetic beads coated with antibody/ligand may be stored at 2-8 degrees C without loss of antigen binding capacity. For long-term storage, a final concentration of 0.02% NaN3 may be added to the antibody-coupled beads in a physiological buffer. Please note that not all coupled antibodies retain their function in long term storage. Verify your coupled antibody stability by testing in small scale. After storage, coated Dynabeads magnetic beads should be washed once in PBS/BSA for 5 min before use.
Find additional tips, troubleshooting help, and resources within ourProtein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Can I perform flow cytometry analysis on cells labeled with Dynabeads beads?
No, the data are not ideal when cells have beads attached to them. You need to use one of the Dynabeads Assays that allow you to remove the beads from your cells for downstream analysis. These include the Dynabeads FlowComp assays and DETACHaBEAD assays.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
