CELLection™ Epithelial Enrich Dynabeads™ - FAQs

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.

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.

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.

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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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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.

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Bead-free cells can be isolated using Dynabeads kits that include DETACHaBEAD technology. These include the Dynabeads FlowComp kits and Dynabeads CELLection Cell Isolation Kits. Also, any of the negative isolation kits will generate cells that are bead-free.

Human Cells
Dynabeads FlowComp Human CD4 Kit (T cells) (Cat. No. 11361D)

Dynabeads FlowComp Human CD8 Kit (T cells) (Cat. No. 11362D)Dynabeads FlowComp Human CD3 Kit (T cells) (Cat. No. 11365D)

Dynabeads FlowComp Human CD14 Kit (monocytes) (Cat. No. 11367D)

Dynabeads CD34 Positive Isolation Kit (hematopoietic progenitor cells) (Cat. No. 11301D)

Dynabeads CD4 Positive Isolation Kit (T cells) (Cat. No. 11331D)

Dynabeads CD8 Positive Isolation Kit (T cells) (Cat. No. 11333D)

Dynabeads Regulatory CD4+/CD25+ T Cell Kit (Cat. No. 11363D)

DETACHaBEAD CD19 Kit (Cat. No. 12506D)

CELLection Epithelial Enrich Dynabeads (Cat. No. 16203)

Dynabeads Human Dentritic Cells (DC) Enrichment Kit (Cat. No. 11308D)

Mouse Cells

Dynabeads FlowComp Mouse CD4 Kit (T cells) (Cat. No. 11461D)

Dynabeads FlowComp Mouse CD8 Kit (T cells) (Cat. No. 11462D)

Dynabeads FlowComp Mouse CD4+CD25+ Treg Cells Kit (Cat. No. 11463D)

Dynabeads FlowComp Mouse Pan T (CD90.2) Kit (Cat. No. 11465D)

Dynabeads Mouse Pan T (Thy1.2) (Cat. No. 11443D)

DETACHaBEAD Mouse CD4 Kit (T cells) (Cat. No. 12406D)

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The M stands for magnetic. M-280 refers to hydrophobic 2.8 micron beads, while M-270 refers to hydrophilic 2.8 micron beads. MyOne refers to 1 micron beads.

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Answering this question is not straightforward. It will depend on the detection method. When using HRP (horseradish peroxidase)-based detection system or radioactivity in combination with a good antibody, very little target is required. More target is required when using an AP (alkaline phosphatase)-based detection system. When a sensitive detection system is used, detection will most likely be in the nanogram range. In some cases, pictograms of target can be detected.

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Within practical limits, the elution volume can be scaled up or down to suit your experiment. However, volumes less than 10 µL become more difficult to work with. In addition, the amount of target is important. If you have a lot of beads with a lot of bound target in a small elution volume, your elution may not be very efficient. Typically, 15-100 µL of beads may be eluted in 30 µL. For efficient recovery of the antigen and/or binding partners, the elution volume should at minimum equal the volume of the beads.

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There are several methods to quantify the amount of antibody bound to the beads. The crudest method is to measure the concentration of antibody in the coupling reaction before and after antibody attachment. Either fluorescence measurements or absorbance at 280 nm can be used. Alternatively, you could measure the amount of antibody bound to the beads by fluorescence, chemiluminescence, or radiolabeling detection methods.

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Incubation time will depend on the immunogenicity of the primary antibody and its binding affinity with the specific antigens. For a good primary antibody, 30-40 minutes incubation should work well. If you are working with a poor antibody or a very low-abundance protein, you could try to increase binding by incubating overnight. However, this also increases the chance of background protein binding.

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If the target protein has the same molecular weight as the heavy or light chain antibody, we would recommend covalently binding the antibody to the bead surface. This can be done by either crosslinking the antibody to the Dynabeads Protein A or G magnetic beads, or secondary coated beads, or by using one of the surface-activated Dynabeads magnetic beads.

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Using Dynabeads magnetic beads for protein isolation provides several advantages:

-Rapid binding kinetics: since the number of beads per volume for Dynabeads is approximately 1,000 times higher than for the same volume of a Sepharose slurry, the probability for Dynabeads magnetic beads to hit the target is far greater.

-Incubation time: due to the rapid binding kinetics, the protocol is usually very short.
-Low background: due to the rapid binding kinetics and the short incubation time, the background is also very low.
-Trapping of impurities: the beads offer no internal volume for binding or trapping of impurities.
-Low antibody consumption: this is because Dynabeads magnetic beads are nonporous, uniform superparamagnetic, monodispersed, highly crosslinked polystyrene microspheres consisting of an even dispersion of magnetic material throughout the bead. The beads are coated with a thin layer of a highly crosslinked polystyrene shell that encases the magnetic material and prevents any leakage from the beads or trapping of ligands in the bead interior. The outer layer also provides a defined surface area for the adsorption or coupling of various molecules such as antibodies. Uniformity of bead size and shape provide consistent physical and chemical properties. These uniform physical characteristics lead to high-quality, reproducible results.
-Reproducibility: due to easier practical handling, such as pipetting. No centrifugation steps or preclearing are required.

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No. Not only is dithionite a reducing agent, but the strong affinity of the dithionite ion for bivalent and trivalent metal cations (M2+, M3+) allows it to enhance the solubility of iron, making it a chelating agent. As a result, the iron in the Dynabeads magnetic beads is reduced and pulled out when they are exposed to dithionite. The same is observed if Dynabeads magnetic beads are exposed to DTT and EDTA. With EDTA, we highly recommend checking the minimal amount of EDTA that your specific molecules would tolerate for binding to the Dynabeads, and if it will affect your specific application. For some applications, low concentrations of EDTA can be tolerated by Dynabeads. On the other hand, using 10 mM EDTA with heating affects the binding of biotin molecules to Dynabeads streptavidin.

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Yes, they are compatible with 6-8 M Urea when used during post-coupling steps.

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Dynabeads magnetic beads, being magnetic in nature are really not designed to be centrifuged. That being said, the beads themselves are compact, as the pores in the polymer matrix are filled with magnetic material and coated with a final outer polymer shell that will further add to the rigidity of the beads. Hence, pressure should theoretically not be a problem for the beads themselves, but the force exerted by the beads on surrounding cells in the pellet may be detrimental to the cells.

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Magnetic beads, unlike agarose beads, are solid and spherical, and antibody binding is limited to the surface of each bead. While magnetic beads do not have the advantage of a porous center to increase the binding capacity, they are significantly smaller than agarose beads (1 to 4 µm), which collectively gives them adequate surface area-to-volume ratios for optimum antibody binding.

High-power magnets are used to localize magnetic beads to the side of the incubation tube and out of the way to enable cell lysate aspiration without the risk of also aspirating immune complexes bound to the beads. Magnetic separation avoids centrifugation, which can break weak antibody-antigen binding and cause loss of target protein.

However, an issue with the use of magnetic beads is that bead size variations may prevent all beads from localizing to the magnet. Additionally, while immunoprecipitation using agarose beads only requires standard laboratory equipment, the use of magnetic beads for immunoprecipitation applications requires high-power magnetic equipment that can be cost-prohibitive. Read more about our Magnetic Immunoprecipitation Products (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/immunoprecipitation.html#products).

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This depends on the bead size. In general, for M-450 Dynabeads, 1 mg/mL beads is approximately equivalent to 1.3 X 10E7 beads/mL; for M-280 Dynabeads, 1 mg/mL beads is approximately equivalent to 6.7 X 10E7 beads/mL.

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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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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.

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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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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

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The beads in the CELLection Epithelial Enrichment kits are coated with Ber-EP4, which binds with high affinity to the epithelial cell marker EpCAM (Epithelial Cell Adhesion Molecule). The Epithelial Enrich product does not positively isolate circulating tumour cells, but rather provides the scientist with a highly sensitive enrichment technique that can be applied directly to whole blood, bone marrow, or MNC samples. The product is based on the theory that in disease-free (or rather circulating tumor cell-free) individuals, epithelial cells are normally absent from the blood stream. The isolation of epithelial cells from blood samples is thus a good indication of circulating tumor cells, but this will have to be confirmed by downstream assays. In addition to epithelial cells, a number of leukocytes will also be isolated due to trapping or unspecific binding. These are, however, significantly smaller than the epithelial cells, and researchers in this field should easily be able to visually identify or distinguish epithelial (tumor) cell from these other cells co-isolated with the Epithelial Enrich beads. Some 10,000 or more leukocytes may be isolated along with your epithelial cells from a 5 mL blood sample. The isolated cells should then be subjected to a downstream investigation to (1) verify epithelial cells (e.g., immune-staining via cytokeratin or similar), followed by (2) further analysis of the enriched cells for tumour cell confirmation and characterization.

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The EpCAM antigen is an epithelial cell adhesion molecule that is abundantly expressed both on the majority of cells of epithelial origin, as well as epithelial-derived tumor cells. The Ber-EP4 (anti-EpCAM) antibody on the beads will thus recognize both normal and cancerous epithelial cells, with the assumption that no epithelial cells are normally present in circulating blood when isolating tumor cells from blood, mononuclear cell, or bone marrow samples.

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There are three methods to remove the Dynabeads magnetic beads from the isolated cells:

1. DETACHaBEAD Kits (positive isolation) - where the release agent is a polyclonal anti-Fab reagent outcompeting the binding of the bead-bound antibody on the cell, giving both antibody- and bead-free cells. Kits are available for Human CD4+ and CD8+ T cells, CD19+ B cells, and CD34+ hematopoietic stem cells.
2. CELLection Kits - where the release agent is a DNase enzyme, digesting the DNA-linker between the antibody and the bead, ultimately leading to bead-free cells. Kits are available for human EpCAM (Ber-EP4) epithelial cells and streptavidin (binding any biotinylated antibodies).
3. FlowComp Kits - where the release reagent is biotin that is out-competing the des-biotinylated antibody coupled onto the beads. Available for human and mouse whole T cells, the T cells subsets CD4+ and CD8+, and human monocytes.

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For positive cell isolation we recommend that you use 1-2 x 10E7 total PBMC per mL, and 1 x 10E7 Dynabeads magnetic beads per mL of sample. Another way of determining how many beads you need is to assume approximately 4 beads per target cell. If you want to deplete your sample of a certain target cell, we recommend using the same cell concentration as above (1-2 x 10E7 total PBMC per mL), but at least double the amount of beads to 2 x10E7 Dynabeads magnetic beads per mL of sample. Or alternatively, assume about 8-10 beads per target cell to be depleted.
Note: Most Dynabeads magnetic beads for cell separations are supplied at approximately 4 x 10E8 beads/mL, thus 1 x 10E7 beads is only 25 µL (please check the specific product manual or label for each product to confirm)

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The major advantages of using positive isolation are the high purity and yield. In addition, the initial sample prep is generally not required, since the cells can be isolated directly from any sample such as whole blood, bone marrow, or tissue digests. If using the primary coated beads without bead release, the procedure is very simple and fast; for example you can get isolation of cells directly from whole blood in as little as 25 minutes, which also leads to very high cell viability. A general disadvantages for any positive isolation method is a potential activation of the cells, depending on the function of the target epitope on the target cell. Note that the Dynabeads magnetic beads are not directly causing activation; it is the antibody-cell epitope interaction that causes this. When using positive isolation with a release mechanism, some loss of yield must be expected, since not all links between the cells and beads will be broken.

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Positive isolation is a method for separation of one specific target cell population from a sample. By using Dynabeads magnetic beads, magnetic particles coated with target-specific antibodies, the beads will bind only the cells with the matching surface antigen expressed on the target cells' surface. Two different Dynabeads magnetic beads - based positive isolation methodologies may be employed depending on the downstream assays. Since the beads themselves are quite large (from 1 µm to 4.5 µm in diameter) we don't recommended that you use bead-bound cells directly in any flow cytometer. Thus, if you have a cellular downstream assay and want to use a flow cytometer, we recommend using one of our positive isolation kits where a bead-release mechanism is available.

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No, the PBS must be Ca²⁺- and Mg²⁺-free as these divalent ions can lead to activation of complement and aggregation of cells. Aggregation of cells in the sample can severely reduce both yield and purity of the isolation. EDTA must also be added to the isolation buffer to minimize activation of complement and aggregation. Alternatively EDTA can be replaced by 0.6% sodium citrate.

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When cell purity is the most important criteria, target cell activation is not a concern, or when the downstream applications are isolation of RNA or gDNA, then positive isolation is recommended. When the most important criteria is to isolate cells with minimum disturbance, and where the downstream applications include cell culture, study cell function, morphology, and flow cytometry, then negative isolation is recommended (note that positive isolation of cells can also be used when the downstream applications include cell culture, flow cytometry, or studying cell function, but then a positive isolation with bead release from the cells are required, by using e.g., FlowComp products or the Positive Isolation Kits containing DETACHaBEAD products).

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Thaw cells in their cryovial in a 37 degrees C water bath until a small ice-clump is left. Transfer the cells gently to a fresh 10-15 mL tube immediately after the cells are thawed and add 10 mL 20% FCS/human serum in droplets to the cells while gentle pipetting. Avoid air bubbles. Work fast. Centrifuge the cells 200 X g, 8 minutes. Discard the supernatant. Resuspend in the appropriate buffer/media.

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In general, freezing medium (10% DMSO and 90% FCS) or Gibco Recovery Cell Culture Freezing Medium (Cat. No. 12648-010) work well. Some cells will always die during the freezing process. In addition, freezing and thawing will cause some cells to lyse. The protocol to freeze mammalian cells using Gibco Recovery medium is as follows:

1. Thaw Recovery Cell Culture Freezing Medium, mix well, and keep at 2-8 degrees C until use.
2. For suspension cells proceed to step 3. For adherent cells, gently detach cells from the substrate on which they are growing using a suitable dissociation reagent such as Gibco TrypLE reagent. Resuspend cells in the complete medium required for that cell type.
3. Transfer cell suspension to a sterile 15 mL centrifuge tube.
4. Determine the viable cell density and percent viability using a Countess Automated Cell Counter (similar automated or manual methods may be used) and calculate the required volume of Recovery Cell Culture Freezing Medium to give a final cell density of 1 X 10E6 to 1 X 10E7 cells/mL.
5. Centrifuge cell suspension at 100-200 x g for 5-10 minutes. Aseptically decant supernatant without disturbing the cell pellet. Note: Centrifugation speed and duration may vary depending on cell type.
6. Resuspend the cell pellet in (2- 8 degrees C) chilled Recovery Cell Culture Freezing Medium at recommended viable cell density for specific cell type (typically 1 X 10E6 cells/mL or greater).
7. Dispense aliquots of cell suspension (mix frequently to maintain a homogeneous cell suspension) into cryovials according to the manufacturer's specifications (i.e., 1.5 mL in a 2 mL cryovial).
8. Achieve cryopreservation in an automated or manual controlled rate freezing apparatus following standard procedures (approximately 1 degree C decrease per minute).
9. Transfer frozen cells to liquid nitrogen, (vapor phase); storage at -200 degrees C to -125 degrees C is recommended.

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Bone marrow needs to be washed and diluted prior to addition of Dynabeads magnetic beads to make the sample less viscous. Washing and DNase treatment is recommended for preparing bone marrow cells prior to cell isolation using Dynabeads magnetic beads:

- Mix 2 mL (10E7-10E8 cells) bone marrow with 2 ml PBS w/ 0.1% BSA + 0.6% Na-citrate.
- Centrifuge at 600 g for 8 min at 18-25 degrees C.
- Discard the supernatant and resuspend to 5 mL with PBS w/ 0.1% BSA + 1mM CaCl₂ + 0.5 mM MgCl₂.
- Add 600 Kunitz units DNase I (120 Kunitz units DNase I per milliliter).
- Incubate cells for 30 minutes at 18-25 degrees C with both gentle tilting and rotation.
- Centrifuge cell suspension for 8 minutes, 600 x g, at 18-25 degrees C.
- Discard supernatant and resuspend cell pellet in 5 mL PBS w/ 0.1% BSA.
- Centrifuge cell suspension for 8 minutes, 600 x g, at 18-25 degrees C.
- Discard supernatant and resuspend at 1 x 10E8 cells per milliliter in RPMI 1640 / 1% FCS

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Follow standard tissue preparations using enzymes and mechanical disruption to get a single-cell suspension. Eliminate large aggregates by sieving the digested cell suspension through a cell strainer or filter through a 30 µm filter. Disruption of tissue normally results in some cell death and release of DNA. Free DNA will impair cell capture, recovery, and purity. DNase I treatment is performed by incubating the cell suspension in PBS with 0.1% BSA + 1 mM CaCl₂ + 0.5 mM MgCl₂ and 120 Kunitz units DNase I per ml at 18-25 degrees C for 30 min. (For CELLection products, wash cells to remove DNase before adding the beads.)

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Mononuclear cells (MNC), also known as peripheral blood mononuclear cells (PBMC), are prepared from whole blood, buffy coat, bone marrow, or umbilical cord blood by density gradient separation. The following protocol can be used for standard MNC preparation for positive isolation or depletion protocols:

1. Collect blood sample with anticoagulant present (EDTA, ACD, heparin). Dilute peripheral blood 1 + 1, buffy coat 1 + 2, bone marrow 1 + 1 and umbilical cord blood 1 + 3 in PBS w/ 0.1% BSA + 0.6% Na-citrate or 2 mM EDTA.
2. Layer up to 35 mL of the diluted sample over 15 mL gradient medium (such as Ficoll or Lymphoprep solution) in a 50 mL tube.
3. Centrifuge for 400 x g for 30-40 minutes at 18-20 degrees C. If blood has been stored for more than 2 hours, increase centrifugation time by 10 min.
4. Collect MNC from the interface and transfer cells to a 50 mL tube.
5. Wash MNC three times with PBS w/0.1% BSA by centrifugation at 300 x g for 8 min at 2-8 degrees C.
6. Resuspend the cells to 1 x 10E7 cells per milliliter in PBS with 0.1% BSA and cool to 2-8 degrees C.
Note: MNC contain T cells (50%), B cells (5-10%), NK cells (5-10%), and monocytes (30%) without granulocytes and very few platelets.

For use with Untouched/negative isolation kits, the following protocol is recommended to obtain MNC prep with low platelet numbers and the highest possible purity:

Whole blood/buffy coat and bone marrow can be used as a starting material.

1. Dilute 10-18 mL blood/buffy coat with PBS w/ 0.1% BSA + 0.6% Na-citrate or 2 mM EDTA to a total volume of 35 ml at 18-25 degrees C.
2. Add the diluted blood/buffy coat on top of 15 mL of gradient medium (such as Lymphoprep or Ficoll solution).
3 .Centrifuge at 160 x g for 20 min at 20 degrees C. Allow to decelerate without braking.
4. Remove 20 mL of supernatant to eliminate platelets.
5. Centrifuge at 350 x g for 20 min at 20 degrees C. Allow to decelerate without braking.
6 .Recover MNC from the plasma/Lymphoprep solution interface and transfer the cells to a 50 mL tube.
7. Wash MNC once with PBS w/ 0.1% BSA by centrifugation at 400 x g for 8 min at 2-8 degrees C.
8. Wash MNC twice with PBS w/ 0.1% BSA by centrifugation at 225 x g for 8 min at 2-8 degrees C and resuspend the MNC at 1 x 10E8 MNC per milliliter in PBS w/ 0.1% BSA.

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Buffy coat, also known as leukocyte concentrate, is the middle fraction of an anti-coagulated blood sample that sits under the plasma and on the top of red blood cells after centrifugation of the sample without using a density gradient reagent such as Ficoll solution. Buffy coat contains both leukocytes and platelets and can be used as a source of this cellular material.

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Typically, one milliliter of adult human blood contains:
~5 x 10E9 red blood cells
~7 x 10E6 leukocytes
~3 x 10E8 platelets

In the 7 x 10E6 leukocyte fraction, there are:
4 x 10E5 monocytes
1 x 10E5 NK cells

Lymphocytes:
2 x 10E5 B cells
1 x 10E6 T cells (approx. 70% are CD4+ T cells and 30% are CD8+ T cells)

Granulocytes:
5 x 10E6 neutrophils
2 x 10E5 eosinophils
4 x 10E4 basophils


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This will depend on your application. As a guideline, the 4.5 micron beads are best used for cell isolation and activation/expansion. These larger beads have a higher magnetic mobility, they are roughly the same size as mammalian cells, and are less likely to be taken up by the cells. The smaller 1 micron beads and 2.8 micron beads are often used when isolating nucleic acids or proteins, or for immunoprecipitation. In negative cell isolation kits, one micron beads are often used because of their higher binding capacity per milliliter of beads and faster binding kinetics. With negative selection, cells taking up any beads will not be a problem as you want to look at the remaining cell population anyway. The 2.8 micron Dynabeads magnetic beads, coated with secondary antibodies, protein A or protein G, or streptavidin are also used for positive cell isolation with primary antibodies of your own choice, targeting specific cell-surface antigens.

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Three different sizes of Dynabeads magnetic beads are available: One micron beads (look for MyOne magnetic beads in the product name), 2.8 micron beads, and 4.5 micron beads. In general, the binding capacity per milliliter of beads and binding kinetics increases as the bead size reduces.

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Dynabeads magnetic beads are super-paramagnetic, meaning they only display magnetic characteristics when a magnet is present. As soon as the magnet is removed, the beads handle like a liquid and are easily dispersed in the sample tube. For cell isolation purposes, this has clear advantages as it allows for gentle handing and reduced stress to the cells. Secondly, the beads all have the same size and shape, with rapid liquid-phase reaction kinetics. The smooth surface of the beads results in less non-specific binding. These properties tend to reduce variability and allow you to get more reliable and reproducible results for your purifications and your analyses whether you are looking at cells or any other target molecule (RNA/DNA/proteins/protein complexes/organelles/exosomes etc.)

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Here are some suggestions:

- Make sure that pH of the RPMI + 1% FBS is not too high: DNase I works best between pH 7.0-7.4 (O₂ exposure of RPMI will result in increased pH and the pH-indicator will turn blue).
- Do not use RPMI + 10% FBS during DNase I treatment. 10% FBS gives a lower cell yield than 1% FBS (might be caused by DNase I inhibitory factors in some batches of FBS).
- Make sure that the buffer contains Mg²⁺/Ca²⁺ required for DNase activity.
- DNase I must be treated gently. Vigorously stirring of the DNase solution can reduce the enzyme activity. Never vortex the DNAse solution.
- When selecting low numbers of cells, we recommend that you pre-coat tubes with RPMI + 10% FBS to reduce cell loss (cells are sticky and will easily attach to the tube wall during selection).
- DNase I has good activity at 20 degrees C, however, we recommend pre-warming the RPMI + 1% FBS to 37 degrees C before starting DNase I treatment because ‘room temperature' varies from lab to lab.
- After cells are incubated with DNase Releasing Buffer it is essential that the bead-cell complexes are thoroughly pipetted before the magnetic separation to provide mechanical disruption to the DNA linker. Failure to pipette the cells will affect cell yield.

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Here are some suggestions:

- Since the DSB-X biotin -streptavidin bond becomes stronger over time, do not increase the time of the release step longer than stated in the protocol. In some instances a shorter incubation time can lead to higher yield.
- After incubation with the releasing agent, we recommend that you resuspend the sample well by pipetting >10 times using a 1 mL pipette before adding the sample to the magnet.

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Here are some suggestions:

- Sample preparation is very important. All isolations must be performed on single-cell suspensions. For isolation of certain cell types (e.g., monocytes) directly from whole blood, we recommend that you wash the blood before isolation to remove interfering factors in the sample.
- An appropriate mixer must be used for all incubations where ‘mixing' is specified. Any mixer providing either tilting and rotation or end-over-end mixing can be used (e.g., HulaMixer Sample Mixer (Cat. No. 15920D)).
- If using FlowComp kits where the antibodies are added to the cells before adding the beads (indirect method), excess antibodies should be removed by washing prior to adding the beads.
- After incubation with the releasing agent, we recommend that you re-suspend the sample well by pipetting >10 times using a 1 mL pipette before adding the sample to the magnet.
- The recommended isolation buffer must be used for cell isolations. The PBS must be Ca²⁺ and Mg²⁺ free as these divalent ions can lead to activation of complement and aggregation of cells.
- Aggregation of cells in the sample can severely reduce both yield and purity of the isolation.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.