Dynabeads Cell Isolation and Expansion Support—Getting Started

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.) 

Three different sizes of Dynabeads magnetic beads are available: One micron beads (look for Invitrogen 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.

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.

The density of Dynabeads magnetic beads is a challenging property to determine. The reason is that Dynabeads magnetic beads have a 17-37% magnetic iron oxide content in order to have a reasonable magnetic separation time, and the density of the iron oxide is about 4.9 g/cm³. Dynabeads magnetic beads are composite materials, being a mix of polymers and iron oxide, and there are very few polymers that have a density below 1.

The sedimentation rate depends on the bead diameter squared, so the sedimentation of a 1 µm bead is much slower than that of 2.8 µm. The effect of diameter on sedimentation rate is to some extent counteracted by the fact that smaller beads need to have a higher content of iron oxide for magnetic separation applications. Typically, our M-280 Dynabeads (diameter 2.8 µm) have a density of 1.4 g DS/cm³ (DS = dry substance), our M-270 Dynabeads (diameter 2.8 µm) and M-450 Dynabeads (diameter 4.5 µm) have a density of 1.6 g DS/cm³, and our MyOne Dynabeads (diameter 1 µm) have a density of 1.8 g DS/cm³.

Typically, one milliliter of adult human blood contains: 

~5 x 10E9red blood cells 

~7 x 10E6leukocytes

~3 x 10E8platelets 

In the 7 x 10E6leukocyte fraction, there are: 

4 x 10E5monocytes

1 x 10E5 NK cells 

Lymphocytes:

       2 x 10E5 B cells 

       1 x 10E6T cells (approx. 70% are CD4+ T cells and 30% are CD8+ T cells) 

          Granulocytes:

       5 x 10E6neutrophil 

       2 x 10E5eosinophil

       4 x 10E4 basophil

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.

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°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°C.
6. Resuspend the cells to 1 x 10E7 cells per milliliter in PBS with 0.1% BSA and cool to 2–8°C.

As illustrated below, 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°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°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°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°C.
8. Wash MNC twice with PBS w/ 0.1% BSA by centrifugation at 225 x g for 8 min at 2–8°C and resuspend the MNC at 1 x 10E8 MNC per milliliter in PBS w/ 0.1% BSA.

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°C for 30 min. (For CELLection™ products, wash cells to remove DNase before adding the beads.)

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:

1. Mix 2 mL (10E7-10E8 cells) bone marrow with 2 ml PBS w/ 0.1% BSA + 0.6% Na-citrate.
2. Centrifuge at 600 g for 8 min at 18–25 °C.
3. Discard the supernatant and resuspend to 5 mL with PBS w/ 0.1% BSA + 1mM CaCl₂ + 0.5 mM MgCl₂.
4. Add 600 Kunitz units DNase I (120 Kunitz units DNase I per milliliter).
5. Incubate cells for 30 minutes at 18–25°C with both gentle tilting and rotation.
6. Centrifuge cell suspension for 8 minutes, 600 x g, at 18–25°C.
7. Discard supernatant and resuspend cell pellet in 5 mL PBS w/ 0.1% BSA.
8. Centrifuge cell suspension for 8 minutes, 600 x g, at 18–25°C. 
9. Discard supernatant and resuspend at 1 x 10E8cells per milliliter in RPMI 1640 / 1% FCS

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°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 an Invitrogen™ 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 × 10E6 to 1 × 10E7 cells/mL. 
5. Centrifuge cell suspension at 100–200 × 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°C) chilled Recovery™ Cell Culture Freezing Medium at recommended viable cell density for specific cell type (typically 1 × 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°C decrease per minute). 
9. Transfer frozen cells to liquid nitrogen, (vapor phase) storage at –200°C to –125°C is recommended. 

Thaw cells in their cryovial in a 37°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.

We recommend that you select a magnet based on your sample volume:

• Invitrogen™ DynaMag™-2 Magnet (Cat. No. 12321D) magnet working volume: 1 µl–1.5 mL sample volume 
• Invitrogen™ DynaMag™-5 Magnet (Cat. No. 12303D) magnet working volume: 1–5 mL sample volume
• Invitrogen™ DynaMag™-15 Magnet (Cat. No. 12301D) magnet working volume: 4–15 mL sample volume
• Invitrogen™ DynaMag™-50 Magnet (Cat. No. 12302D) magnet working volume: 10–50 mL sample volume
• Invitrogen™ DynaMag™-96 Side Magnet (Cat. No. 123-31D): pulls beads to the side of each well. Compatible with PCR-strips and 96-well plates (200 μL), non-skirted and half-skirted
• Invitrogen™ DynaMag™-96 Bottom Magnet (Cat. No. 123-32D): pulls beads to the bottom of each well. Compatible with PCR-strips and 96-well plates (200 μL) non-skirted and half-skirted

The best way to remove all beads is first to keep the tube longer on the magnet. This is followed by very gentle pipetting of the supernatant over to a new tube (be careful not to touch the beads which are located on both sides of the tube). The supernatant should then be added to the magnet again to remove the remaining few beads.

Spray and/or wipe the DynaMag magnet unit with one of the following cleaning agents:

- 70% (v/v) isopropyl alcohol
- 1% (v/v) sodium hypochlorite solution (bleach)
- 0.1 N HCl solution

Do not submerge the magnet unit in aqueous solutions and avoid prolonged exposure to water or aqueous solutions. Clean with a damp cloth and mild detergent when exposed to harsh solvents. Do not autoclave the magnet unit as heating the magnet to above 70 degrees C can destabilize the magnetic material, resulting in loss of magnetic strength.

Negative isolation, also known as untouched cell isolation, uses an optimized cocktail mixture of antibodies directed toward the different non-target cell types in a sample followed by magnetic bead separation to pull out antibody bound cells and leaves the target cells intact in the supernatant. Dynabeads™ magnetic beads used in the negative isolation are either coated with streptavidin (if the antibodies in the cocktail are biotinylated) or secondary antibodies (if the antibodies in the cocktail are from a single host species). The workflow of negative isolation is illustrated in the figure below:

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).

The advantages of negative isolation are that the isolation procedure is fast, and the isolated cells remain untouched and, therefore, are not likely to be stimulated. Since the method is very gentle, the viability of isolated cells is high. The disadvantages are that negative isolation normally requires PBMC/MNC as a starting material and the purity is not as high as is achieved with positive isolation.

With negative isolation, the target cell purity can be as high as 90%.

2X rosetting, also known as double rosetting, is a technique that enhances the depletion of cells expressing low levels of the target antigens. This approach does not require more beads but it takes 10 minutes longer to perform. Instead of adding all Depletion Dynabeads™ magnetic beads (e.g., 100 μL) to the prepared antibody coated cells, half (50 μL) is added, incubated, and the supernatant (target cells) is transferred to a new tube containing the rest of the Depletion Dynabeads™ magnetic beads (50 µL). Incubate for 10 minutes and transfer the supernatant to a fresh tube. Negative isolation using double rosetting can increase purity.

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.

These products have a recovery rate of 73±10% with 93±3% purity.

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. The workflow for positive isolation kits with bead release is summarized in the figure below (Note: in some of the positive isolation kits the antibodies are directly linked to the Dynabeads™ magnetic beads, so for these kits the first two steps are skipped). On the other hand, if you want to isolate your cells for further downstream molecular studies, you don’t need to release the beads from the cells and can use the direct conjugated beads not containing any release mechanism.

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. 

• 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).

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

• 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.
• 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). 
• 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.

The major advantage using DETACHaBEAD™ Kits is to obtain both antibody-and bead-free cells after isolation. In addition, the purity and viability is high. Unfortunately, DETACHaBEAD™ Kits are available for only a few human cell types—the release cannot be made generic, since there is one unique DETACHaBEAD for each product. To obtain the optimal yield, the release step needs to be performed at room temperature for about 45 min. DETACHaBEAD™ reagents will interfere with the FACS readings of the cells after isolation, so it is important to wash the cells after release according to the protocol.

DETACHaBEAD™ reagents are polyclonal anti-mouse anti-Fab antibodies that bind to the antigen-binding region of the target antibodies with high affinity, thereby reducing their binding affinity to the cell. Each of the DETACHaBEAD™ reagents has been optimized to detach antibodies of one specificity. Therefore, we don’t recommend that you use the DETACHaBEAD™ reagent for any other cell type or Dynabeads™ magnetic beads type than is supplied in the kit. DETACHaBEAD™ products are only suited for detachment of bead-bound antibodies, and not free antibodies bound to cells. 

No, the Pan B antibody coupled onto the Dynabeads™ magnetic beads does not cross react with mouse B cells or B cells from other species. 

Whole blood contains soluble CD14 that binds to the beads, leaving fewer binding sites available for binding to the CD14 antigen on the monocytes and reducing overall monocyte yield.

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.

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.

Dynabeads™ FlowComp™ kits are based on modified biotin-streptavidin isolation and release technology. The capture antibodies are labeled with DSB-X biotin, while the Dynabeads™ magnetic beads in the Dynabeads™ FlowComp™ kits are coated with modified streptavidin. The DSB-X biotin on the target antibody has a lower affinity to the streptavidin than normal biotin. Thus, following the positive isolation, the cells are released by adding the release buffer containing excess of normal biotin that out-competes the binding between the modified streptavidin on the Dynabeads™ magnetic beads and the DSB-X biotin-labeled target antibody. As a result, the isolated cells are bead-free but not antibody-free (the DSB-X biotinylated target antibody is still attached to the cell surface).

We recommend using FACS to check the cell purity. But, since the target antibody is still attached to the cell surface, we recommend that you use an antibody that binds at a different epitope of the same surface marker. In the product insert, we give recommendations for the best suitable antibodies for use in FACS.

Most human FlowComp™ kit protocols are for 5 x 10E7 total cells or more per test. You can work with fewer cells, but we don’t recommend that you scale down the volume of Dynabeads™ magnetic beads or the buffers; stick to the volumes recommended for 5 x 10E7 cells. This is also true for the release step, where the volume of release buffer must be at least 500 μL, so make sure not to scale down more than recommended.

No, it is very important to use the supplied biotinylation module provided in the Dynabeads™ FlowComp™ Flexi kit since the Release Buffer only will out-compete the binding with DSB-X biotin and not ordinary biotin. You will probably get good isolation using your own biotinylated antibody, but you won’t get any release unless you use the DSB-X biotinylated antibodies supplied in the kit.

No, both Biotin Binder and streptavidin Dynabeads™ magnetic beads are coated with wild type streptavidin, which binds DSB-X biotin too tightly to be efficiently released by free normal biotin.

Given that the concentration of your antibodies is low, the biotinylation efficiency will be reduced (the lowest concentration given in the protocol is 0.5 mg per mL). In-house, we have not used less than 0.5 μg per mL of antibody, but increasing the amount of DSB-X biotin (Table 1 in manual for DSB-X Biotin Protein Labeling Kit (Cat. No. D-20655) from 2μL to 4μL in the coupling reaction might increase the efficiency).

Biotinylation of 0.5 mg per mL antibody according to the protocol:
0.5 mg per mL x 0.2 mL = 0.1 mg antibody
Yield of biotinylated antibody: (0.1 mg antibody x 0.85%) in 0.2 mL = 0.4 mg per mL
Amount of biotinylated antibody in 25 μL: 0.4 mg per mL x 25 μL = 10 μg antibody in 25 μL, which is in the recommended range (5-50 μg for 5 x 10e7 cells).

Biotinylation of 0.2 mg per mL: 0.2 mg per mL x 0.2 mL = 0.04 mg antibody
Yield of biotinylated antibody: (0.04 mg antibody x 0.85%) in 0.2 mL = 0.17 mg per mL
Amount of biotinylated antibody in 25 μL: 0.17 mg per mL x 25 μL = 4 μg antibody in 25 μL, which is slightly outside the recommended range.

Note: Antibody may be easily concentrated without affecting the biological activity by using centrifuge concentrators (i.e., Centricon filter columns).

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

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.

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.

Whether cells will internalize the Dynabeads™ magnetic beads during culture will depend on the cell type. Due to the bead size (usually 4.5 μM 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 the answer to your question is both yes and no— depending on the cell type.

Exosomes are small, membrane-bound ovoid to cup shaped particles around 30–150 nm in size containing mRNA, microRNA, proteins, and lipids. Exosomes are released by normal, abnormal, or neoplastic cells into body fluid such as blood, urine, saliva, and breast milk. Exosomes originate from the endocytic compartment and are released from cells as multivesicular bodies (MVB) fused with plasma membrane (J Cell Biol 200:373 (2013)).

A range of different functions have been reported such as antigen presentation, apoptosis, angiogenesis, inflammation, and coagulation by protein/lipid exchange or activation of a signaling pathway. Exosomes provide a novel vehicle for genetic exchange between cells and mediate cell to cell communication. Exosomes also transport and propagate of infectious cargoes such as prion and retrovirus. 

Exosomes can be isolated by ultracentrifugation or density gradient separation in addition to a precipitation approach. Exosomes can also be isolated by a magnetic approach using Dynabeads™ magnetic beads targeting exosome markers such as Human CD9, CD63, CD81, EpCAM or secondary antibody–coated Dynabeads™ magnetic beads using different antibodies against other exosomal surface markers.

It depends on the cell source from which the exosomes were derived. The most commonly used surface markers for isolating and characterizing exosomes are CD9, CD63, CD81, or TSG101. The following table lists some of the recent references and surface markers for identifying or isolating exosomes:

Exosomes are usually characterized by flow cytometry (using surface markers such as CD9, CD63, TSG101, and Alix), by EM to study morphology and size, or by detailed protein analysis by LC-MS/MS. 

We have exosome isolation kits for Exosome-Human CD63 (Cat. No. 10606D), Exosome-Human CD9 (Cat. No. 10614D), Exosome-Human CD81 (Cat. No. 10616D), and Exosome-Human EpCAM intended for isolating exosomes with these commonly used exosome surface antigens. If you are interested to isolating exosomes with other specific surface markers using your own antibody, you can use our Dynabeads™ exosome immunoprecipitation (Protein A, Cat. No. 10610D), Dynabeads™ exosome immunoprecipitation (Protein G, Cat. No. 10612D), or Exosome-Streptavidin for isolation/detection (Cat. No. 10608D). In addition, anti-mouse IgG Dynabeads™ magnetic beads (Cat Nos. 11031 or 11033) also can be employed in exosome isolation using mouse monoclonal antibodies against selected surface markers. These products are summarized in the table below:

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.

The M-450 Tosylactivated beads (Cat. No. 14013) are very similar in terms of binding chemistry, concentration, and coupling protocol to the discontinued product Dynabeads™ M-500 Subcellular.

This depends on the host of the antibodies you are going to use. Protein A and Protein G differ in their binding strength to antibodies/immunoglobulins (Ig) from different species and subclasses. The following table provides information about the binding strength of protein A or protein G to different types of antibodies:

Yes, exosomes isolated with different surface markers can be distinctive in their protein profile. This has been demonstrated by Tauro et al., who isolated two distinctive populations of exosomes based on surface markers EpCam or A33 from conditioned cell culture medium from a human carcinoma cell line. This proteomics study indicated that these two populations of exosomes are unique.

Yes. See this poster.

In addition, here are some citations:

• Blood 91:2573 (1998)
• Science 289:444 (2000)
• J Physiol 537:537 (2001)
• Mol Cell Proteomics 12:587 (2013)
• Biol Reprod 81:717 (2009) 

In general, all membrane-bound organelles can be isolated using the Dynabeads™ magnetic beads. In combination with your own organelle-specific antibody, the immuno-magnetic isolation approach has several advantages:

• The method is rapid and gentle
• Does not require any labor-intensive density gradient and ultracentrifugation
• Organelle populations with different density can be isolated
• Highly pure organelles are obtained

Several antibodies that recognize antigenic sites on organelles are commercially available. You can choose between two techniques:

• Direct technique: Coat the Dynabeads™ magnetic beads with your antibody/ligand by incubating the two together for a short incubation period. Then mix your coated beads with your crude fraction. Target organelles bound to the Dynabeads™ magnetic beads are separated by the use of a magnet, and washing is performed in an equally fast and easy manner.
  
  
• Indirect technique: Mix the antibody/ligand with the target-containing crude fraction and incubate to allow them bind. Then add the Dynabeads™ magnetic beads to the sample, incubate for a short period to allow the target-antibody/ligand complex bind. Separate and wash using a magnet. The indirect technique cannot be used if the target-specific antibody/ligand is to be coated directly onto one of the surface-activated beads.

Due to the speed and efficiency of isolation, the direct technique is recommended. However if the antigen on the target organelles is inaccessible (e.g., buried within the vesicle), the indirect technique may be advantageous.

It is essential to identify an appropriate antigen and to have a high-affinity antibody that recognizes the antigen in its native, un-denaturated state. The epitope on the subcellular fraction must be free to bind the specific antibody. If the epitope is “buried” within the fraction and not exposed on the surface, it may not be possible to isolate the target organelles.

It is very important to properly homogenize the cells before use. Homogenization may be easier for some samples such as liver, than for other samples such as yeast. If nuclei are broken during homogenization, DNA will be released and may cause clumping of organelles. Organelle clumps in the unpurified cellular material will make the immuno-isolation technique difficult.

Dynabeads magnetic beads coated with antibody/ligand may be stored at 2–8°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.

Yes. We recommend using Dynabeads™ M-270 Epoxy or Dynabeads™ M-450 Epoxy for this purpose as the reaction chemistry of these particles allows the vesicles/organelles to bind at low temperature (4°C). We don’t recommend using Dynabeads™ M-280 Tosylactivated or Dynabeads™ M-450 Tosylactivated as they will require a higher reaction temperature (37°C), and it is usually desirable to keep the vesicles/organelles at low temperature. The purified vesicles/organelles bind covalently to the Dynabeads™ magnetic beads via vesicle/organelle-proteins. We recommend adding a blocking protein (e.g., BSA) after 1 hour of incubation.

If the sample is very viscous, a large bead is preferred (4.5 µm), which has a higher magnetic content and moves more easily to the magnet in very viscous samples. If high binding capacity is preferred, choose the 2.8 µm bead.

This will vary a lot depending on cell type, organelle type and abundance, primary antibody and bead type, it is difficult to give very specific recommendations. Most likely you will need to do a titration of bead-to-organelle ratio to optimize the isolation. Use approximately 2 x 10E7 beads per 100–200 µg of unpurified cellular fraction (lysate) diluted in 300 µL buffer (e.g., PBS w/ 2 mM EDTA and 5% FCS or BSA) as a starting point. If your antibody has high affinity, you should be able to achieve a near-complete selection of your target vesicles. Published articles report from 10–20 (e.g., peroxisomes) to hundreds of organelles bound per bead, but this will depend on your antibody and the coating efficiency of the beads.

Dynabeads™ magnetic beads used for organelle isolation are not designed to elute off the organelles after isolation. When isolating proteins, we recommend using high salt or low pH (boiling in SDS buffer will cause denaturation of the proteins). If you want to try to elute the organelles off, you need to consider the interaction between the organelle and ligand/antibody. If this interaction is strong, you will need more harsh conditions than if the interaction is weaker. High salt may cause the organelles to burst (osmotic stress). You may try low pH, but we can't guarantee the results, and you will need to optimize. We have one product—Dynabeads CELLection™ Pan Mouse IgG (Cat. No. 11531D)—in which a DNA linker connects the antibody (anti-mouse IgG) to the Dynabeads™ magnetic beads, and this linker can be broken after isolation using a DNase, to free the antibody with isolated organelle. Our FlowComp™ Flexi Kit (Cat. No. 11061D) can also be used.

We have several surface-activated Dynabeads™ magnetic beads that allow you to couple your own antibody covalently to the beads. These products include M-450 Tosylactivated beads (Cat. No. 14013), M-280 Tosylactivated beads (Cat. Nos. 14203 and 14204), M-450 Epoxy beads (Cat. No. 14011), M-270 Epoxy beads (Cat. No. 14301), M-270 amine beads (Cat. No. 14308), and M-270 carboxylic acid (Cat. No. 14306D).

If you are not concerned about bead release you can use:

Secondary-coated Dynabeads™ magnetic beads

Dynabeads™ ProteinA/G magnetic beads

Dynabeads™ Streptavidin magnetic beads

If you would like to remove beads gently after isolation you can use:

FlowComp™ Flexi magnetic beads

Dynabeads™ CELLection™ Biotin Binder

Dynabeads™ CELLection™ Pan mouse IgG

Citations for isolation of endosomes and lysosomes:

• Mol Biol Cell 11:2775 (2000)
• J Biol Chem 275:7004 (2000)
• J Cell Sci 111:141 (1998)
• Proc Natl Acad Sci U S A 95:8613 (1998)

Citations reporting the isolation of transport vesicles originating from the ER:

• Science 289:444 (2000)
• Science 295:848 (2002)
• Mol Biol Cell 9:623 (1998)
• J Cell Sci 110:2117 (1997)
• J Neurosci 21:7506 (2001)
• J Biol Chem 274:35577 (1999)

Citations reporting the use of Dynabeads™ magnetic beads to isolate mitochondria and other organelles:

• Electrophoresis 19:1205 (1998)
• Biochem Biophys Res Commun 295:1027 (2002)
• J Neurosci 21:7506 (2001)
• J Biol Chem 276:32338 (2001)
• J Cell Sci 110:2117 (1997)
• Jones SM, et al (1998) In: Cell Biology: A laboratory Handbook. Academic Press. pp 12–25
• J Cell Biol 138:291 (1997)
• J Biol Chem 275:7004 (2000)

Citations reporting the use of Dynabeads™ magnetic beads to isolate mitochondria and chloroplasts and also mitochondrial/chloroplast nucleic acid:

• Genomics 60:67 (1999)
• Biotechniques 26:336 (1999)
• Int J Legal Med 108:237 (1996)
• Am J Hum Genet 60:408 (1997)
• Int J Legal Med 106:85 (1993)
• Biotechniques 16:484 (1994)
• Mol Ecol 4:731 (1995)

In nature, activation of T cells occurs through the engagement of both the T cell receptor (TCR)/CD3 and CD28 on the T cell by the major histocompatibility complex peptide and B7 family members on the antigen-presenting cells (APC) such as dendritic cells. Both of these two stimulating signals are required for production of an effective immune response. The first signal ensures that only a T cell with a TCR specific to that peptide is activated. The only co-stimulatory receptor expressed constitutively by naive T cells is CD28, this second signal licenses the T cell to respond to an antigen. In the absence of CD28 co-stimulation, T cell receptor signaling alone results in anergy. 

Dynabeads™ magnetic beads coated with CD3/CD28 antibodies are artificial APCs (aAPC) and mimic the natural activation of T cells. 

The mechanisms of T cell activation, both in nature (upper figure) and by using Dynabeads™ magnetic beads coated with CD3/CD28 antibodies (lower figure), are illustrated in the figure below:

Dynabeads™ CD3/CD28 offers higher expansion rate and more consistent results than traditional methods (e.g., mitogens or soluble/plate-bound antibodies). This is due to:

• Reproducible coating of optimized antibody ratios on the beads 
• The bead size is close to mammalian cells, thus truly mimicking the APC to provide three-dimensional and simultaneous signals to TCR/CD3 and CD28 on the target T cells
• Dynabeads™ CD3/CD28 offer a ready-to-use format that is simple and easy to use and have 24 months shelf life from the manufacturing date 
• An expansion platform covering mouse and human research, translational research, and clinical applications
• Activates naive T cells into a more central memory phenotype (Cytotherapy 16:619 (2014)).

Bead size for all of the T cell activation and expansion kits is 4.5 μm.

We recommend using AB human serum (serum taken from donors with AB blood type that do not have antibodies against A or B). Regular FBS can also be used, but most people switch to AB serum for T cell clone expansion studies.

Some CD8+ T cells don’t express the CD28 antigen. This means that when working on CD8+ T cell clones, as opposed to CD4+ clones, it is essential to determine if the CD8+ clones are expressing CD28. For polyclonal expansion of CD8+ use the recommended number of beads per cell for activation and re-stimulation as described in the product manual. Do not re-stimulate before day 8–12 depending on the application (very strong cell:bead clusters are formed, and the cells are producing a lot of IL-2, which is important for growth, so the media should not be changed at this point). Another optional recommendation is to use 20–100 U IL-2 in the medium when re-stimulating, but this not critical.

For antigen-specific/primed human CD8+ cells, we recommend using the Dynabeads™ Human T-Activator CD3/CD28/CD137; CD137 helps these cells to grow and expand better and Dynabeads™ magnetic beads coated with CD3 and CD28 antibodies only. Please see the following reference. 

The activation of T cells happens within minutes. When you analyze the cells depends on your assay (e.g., T cell signaling: within minutes, for in vitro expansion: 8–12 days). 

CD4+ T helper cells stimulated with Dynabeads™ CD3/CD28 will differentiate into different T helper subsets, depending on the cytokines added during in vitro culture conditions.

• Dynabeads™ + IL-2 give a Th1 subset (IL-12 may also be added)
• Dynabeads™ + IL-4 + IL-2 give a Th2 subset
• Dynabeads™ +IL-1b + IL-6, IL-21 and TGFβ give a Th17 subset

Dynabeads™ CD3/CD28 is optimized for activation of naive T cells by giving a strong anti-CD3 signal and a co-stimulation signal with anti-CD28 antibody in close proximity. After stimulation, the majority of the T cells are T stem central memory/T central memory cells expressing CD62L, CD45RO, CD28, CD27, CCR7 and CD95.

Only a small fraction of the cells are effector memory T cells or terminal differentiated (T_EMRA CD57+) cells, in contrast to T cells expanded with anti-CD3 antibody (OKT-3).

This depends on the product you are using:

• CTS Dynabeads™ CD3/CD28 (Cat. No.40203D) and Dynabeads™ Human T-Expander CD3/CD28 (Cat. No. 11141D): For T cell expansion (>3 days) use 3 beads per T cell (3:1). For re-stimulation use 1 bead per cell (1:1).
• Dynabeads™ Human and Mouse T Cell Activation CD3/CD28 (Cat. Nos. 11131D, 11132D, 11161D, 11456D, 11452D, 11453D): For short-termstimulation, expansion, and re-stimulation, use 1 bead per T cell (1:1).
• Dynabeads™ Human Treg Expander (Cat. No. 11129D): For expansion use 4 beads per T cell (4:1).
• Dynabeads™ Human T-Activator CD3/CD28/CD137 (Cat. No. 11162D, 11163D): For expansion and re-stimulation of T cell clones/cell lines use 1 bead per 5 - 10 T cells (1:10).

No, only T cells will get the stimulation signal and grow in culture. After a week the purity of CD3+ cells is typically above 95%. Stimulating MNC can be problematic due to the presence of monocytes as they will phagocytose the beads. We recommend that you remove the monocytes from the MNC or to isolate T cells before T cell activation/expansion by standard methods such as freeze/thaw, plastic adherence, or use of Dynabeads™ CD14 (Cat. No. 11149D, for human RUO). 

After 3–4 days in culture, most of the Dynabeads™ magnetic beads have detached from the cells. 

For expansion protocols, we recommend that you remove the beads with the help of a magnet between days 8–12 depending on the assay. Pipette the bead:cell complexes thoroughly before applying to the magnet for 2 minutes, and transfer the supernatant with the released cells to a new tube. Discard the used beads and add fresh beads for re-stimulation.

For short-term activation, the cells can be lysed while they are still on the beads for molecular assays. Between days 1–3, the beads form strong clusters with the cells, thus it is hard to remove the beads in this period without losing a lot of the cells. 

Yes, when T cells are incubated with Dynabeads™ magnetic beads, they typically form visible cell-bead clusters in the wells within 12 hours. After 2–3 days the cells enter the blast stage (cell division) where they increase considerably in size and their morphology changes to a more stretched appearance. After expansion (days 8–12), they will regain their usual morphology of round and clearly defined cells. The picture below shows morphological change with typical appearance of T cells after 2–4 days of expansion of antigen specific CD8+ T cells with Dynabeads™ Human T-Activator CD3/CD28 (Cat. Nos. 11161D, 11131D, 11132D).

No, it does not. It is a specific anti-CD28 antibody.

Generally we recommend re-stimulating with fresh beads between days 8–12 depending on your application. Follow the product manual for instructions on re-stimulation.

Dynabeads™ CD3/CD28/CD137 is optimized for activation of Ag-specific/primed T cells by giving a weak anti-CD3 signal and co-stimulatory signals with the anti-CD28 and anti-CD137 antibodies in close proximity. For activation and expansion of memory T cells we recommend 1 bead per 10 target cells. For expansion of primed/antigen-specific T cells, too many beads per T cell may induce apoptosis. In contrast, the Dynabeads™ CD3/CD28 (without the CD137) products are optimized for activation of naive T cells which require a stronger anti-CD3 activation signal than Ag-specific/primed T cells. 

Note: Specific for Dynabeads™ Human T activator CD3/CD28/CD137 (Cat. Nos. 11162D, 11163D)

The major differences are:

• Intended use: Dynabeads™ Human CD3/CD28/CD137 is optimized for activation of Ag-specific/primed T cells by giving a weak anti-CD3 signal and co-stimulatory signals with the anti-CD28 and anti-CD137 antibodies in close proximity. Dynabeads™ Human T Activator CD3/CD28 (without the CD137) is optimized for activation of naive T cells, which require a stronger anti-CD3 activation signal than Ag-specific/primed T cells. 
• Different clones: The CD3 and CD28 antibodies of these two products are from different clones.
• Different protocols: There are very few beads required when activating Ag-specific/primed T cells (1 bead per 10 target cells) vs. naive T cells (1 bead per target cell), so follow the protocols for optimal results. Due to this, the bead concentration is also lower in the Dynabeads™ CD3/CD28/CD137 product. The solution will appear clear, and not as brown as the other Dynabeads™ magnetic beads, but, there are beads present! Mix the vial properly before taking out the required bead volume for any experiment.

Dynabeads™ Human T-Activator CD3/CD28/CD137 offers a simple method for activation and expansion of antigen-specific/primed T cells that does not require antigen-presenting cells or antigen. There is no need to identify and prepare donor-matched MNC, which saves a lot of time, donor blood, and the use of Ficoll™/Lymphoprep™ solutions and antigen. The mechanism of activating antigen-specific T cells by Dynabeads™ Human T-Activator CD3/CD28/CD137 is summarized in the figure below. 

Dynabeads™ Human T-Activator CD3/CD28/CD137 mimics in vivo T cell activation from antigen-presenting cells by utilizing the three activation signals CD3, CD28 and CD137, bound to a three-dimensional bead similar in size to the antigen-presenting cells.

We recommend using 1:10 bead-to-target cell ratio. Culture the cells in cell culture media of choice (e.g., advanced RPMI-1640/CTS™ OpTmizer™ T Cell Expansion SFM) supplemented with 2% human AB serum with 2 mM of glutamine and 50 U/mL of rIL-2. For expansion of CD8+ T cells we recommend that you add 5 ng/mL of rIL-7. The following table summarizes the seeding density, volume of beads per well, and medium volume.

Yes, all T cells up-regulate CTLA-4 upon activation. However, when using Dynabeads™ magnetic beads for activation of T cells, no ligand for CTLA-4 (B7) is present, so T cell expansion is not inhibited. In contrast, APCs express B7 that binds CTLA-4 and cause an inhibitory signal.

Dynabeads™ Human T-Activator CD3/CD28 is optimized for activation of naive T cells, whereas Dynabeads™ Human Treg Expander is optimized for regulatory T cells. Both products contain the same antibody clones in the same CD3:CD28 ratio.

We offer Dynabeads™ Mouse T-Activator CD3/CD28 in 3 different volume sizes (Cat. No. 11456D (0.4 mL), Cat. No. 11452D (2 mL), Cat. No. 11453D (5 x 2 mL)). This product is intended for activation of mouse CD4+ or CD8+ T cells, or mouse Treg cells. 

We recommend that you culture mouse T cells in advanced RPMI-1640 with 2 mM glutamine and 10% FBS supplemented with 30 U/mL mouse rIL-2 (human rIL-2 can also be used). The optimal bead-to-target cell ratio is 1:1. For short-term activation of mouse T cells, we recommend starting with 8 x 10E4 mouse T cells/well in 100–200 μL medium in 96-well culture plates. For expansion of mouse T cells, we recommend that you start with 1–1.5 x 10E6/mL T cells in suitable culture plate or flask. The bead-to-cell ratio and concentration of IL-2 is the same as for activating T cells.

We recommend that you culture mouse T cells in advanced RPMI-1640 with 2 mM glutamine and 10% FBS supplemented with 2000 U/mL mouse rIL-2 (human rIL-2 could also be used). The bead-to-cell ratio is 2:1. We recommend that you start with 1–1.5 X 10E6/mL T cells in suitable culture plate or flask for expanding mouse Treg cells.

The rule of thumb is to seed the T cells at a concentration of ~0.5–1 x 10E6 cells/mL and to split the T cells when the concentration is ~1.5–2.5 x 10E6 cells/mL.

We recommend the following cell numbers and volumes for these plates:

In nature there is a constitutive internalization and re-entry of the TCR-CD3 complex to the cell surface. When Dynabeads™ magnetic beads bind to CD3/CD28 on the cells in culture, the TCR-CD3 complex is internalized and then degraded. When this happens the beads do not have any target on the surface of the cells to bind to and therefore the beads will be released into the medium. The bead-cell clusters last for about 3–4 days until the beads fall off the cells.

The T-cell receptor (TCR-CD3) complex consists of several different subunits where the variable immunoglobulin domains of TCR (V) bind to the ligand. The cytoplasmic tails of the CD3 subunits and TCR interact with cytosolic signaling proteins. In this cytosolic part of the complex several types of internalization signals are located.

Constitutive internalization (without ligand binding):

From the literature we understand that approximately 20 tyrosine-based internalization motifs and 2 di-leucine motifs will constitutively remove the TCR- CD3 complex from the plasma membrane without ligand binding. The recycling time is approximately 100 minutes (from plasma membrane to early endosomes and back to plasma membrane), and after 10 such cycles the TCR- CD3 complex is targeted for degradation in lysosomes.

Induced internalization (with ligand binding):

The ligand-dependent internalization of the TCR-CD3 complex is a ubiquitin-dependent internalization process. Upon ligand binding, ubiquitin is coupled to a certain amino acid in the cytosolic tail of the complex, which results in a clathrin-dependent internalization of the complex followed by lysosomal degradation (the internalization reaches plateau around 2 hours (Nature 375:148 (1995)) 

When the beads bind to CD3-CD28, the complex will actively be internalized and degraded. When this happens the beads do not have any target on the surface of the cells to bind to and therefore the beads will be released into the medium. If the beads are not removed from the medium they can re-bind to the TCR- CD3 complex when they re-appear at the plasma membrane.