Product FAQ

Do you have any references for isolation of mice endothelial cells using Dynabeads magnetic beads?

Answer

These are several references describing isolation of endothelial cells from various murine samples or tissues using different endothelial markers. In addition, we have included a brief description of the Dynabeads product used and the epithelial cell target antigen for each of the references.

Carilo A et al. (2004) Transcription regulation of TNF-alpha-early response genes by poly(ADP-ribose) polymerase-1 in murine heart endothelial cells. Nucleic Acids Res 32(2): 757-766.
Mouse heart endothelial cells were isolated using rat anti-mouse CD31 (clone MEC13.3, BD Pharmingen) and Dynabeads M-450 sheep anti-rat IgG.

van Wijngaarden J et al. (2004) Identification of differentially expressed genes in a renal cell carcinoma tumor model after endostatin-treatment. Lab Invest 84:1472-1483.
Mouse endothelial cells were isolated using rat anti-mouse CD31 (clone ER-MP12) and Dynabeads M-450 sheep anti-rat IgG.

Kataoka H et al. (2003) Protease-activated receptors 1 and 4 mediate thrombin signaling in endothelial cells. Blood 102(9):3224-3231.
Mouse endothelial cells were isolated using Dynabeads M-450 sheep anti-rat IgG and a rat anti-mouse ICAM-2 (CD102) antibody from BD Pharmingen.

Lim YC et al. (2003) Heterogeneity of endothelial cells from different organ sites in T-cell subset recruitment. Am J Pathol 162(5):1591-1601.
Dynabeads sheep anti-rat IgG were coated with either anti-PECAM-1 (CD31) or anti-ICAM-2 (CD102) monoclonal antibody (2.5 µg antibody for 2 x 10e7 beads) and used for isolation of mouse lung or heart endothelial cells.

Camerer E et al. (2002) Genetic evidence that protease-activated receptors mediate factor Xa signaling in endothelial cells. J Biol Chem 277(18):16081-16087.
Mouse ECs isolated using Dynabeads magnetic beads coated with a rat anti-mouse ICAM-2 (CD102) antibody (BD Pharmingen). Purity was more than 90%.

Armstrong LC et al. (2002) Thrombospondin 2 inhibits microvascular endothelial cell proliferation by a caspase-independent mechanism. Mol Biol Cell 13(6):1893-1905.
Mouse lung epithelial cells were isolated using Dynabeads sheep anti-rat IgG and a BD Pharmingen rat anti-mouse ICAM-2 (CD102) antibody.

Tiruppathi C et al. (2002) Impairment of store-operated Ca2+ entry in TRPC4(-/-) mice interferes with increase in lung microvascular permeability. Circ Res 91:70-76.
Mouse lung endothelial cells were labelled with a rat anti-mouse PECAM-1 (CD31) antibody (BD Pharmingen), and endothelial cells isolated using the Dynabeads M-450 sheep anti-rat IgG. Isolated cells were washed, detached from the beads using trypsin treatment for 3 minutes, and put in culture.

Yeh JR et al. (2000) The antiangiogenic agent TNP-470 requires p53 and p21CIP/WAF for endothelial cell growth arrest. Proc Natl Acad Sci 97(23):12782-12787.
Dynabeads M-450 sheep anti-rat IgG and rat anti-mouse E-selectin (CD62E, BD Pharmingen) were used for isolation of mouse pulmonary (lung) endothelial cells.

Answer Id: E6143

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Product FAQ

How can I remove the Dynabeads magnetic beads from the cells isolated, and what are the major differences between the bead releasing kits?

Answer

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.

Answer Id: E12102

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Product FAQ

Can Dynabeads magnetic beads be used for viral isolation?

Answer

Theoretically, virus isolation works the same way as cell isolation, organelle isolation, or protein isolation. We don't have any protocols for virus particle isolation, but different viruses (e.g. hepatitis A, B, and C particles) have been isolated using Dynabeads magnetic beads.

First, you need some kind of target-specific ligand (e.g. an antibody) and this ligand can be coupled to Dynabeads magnetic beads in different ways, either using surface-activated beads (e.g Dynabeads M-280 Tosylactivated), beads coated with secondary antibody (such as Dynabeads M-280 Sheep anti-Rabbit IgG or Dynabeads Sheep anti-Mouse IgG), or beads coated with Protein A, Protein G, or streptavidin (e.g. Dynabeads M-280 Streptavidin, which is a good choice if you have a biotinylated specific antibody).

For antibody immobilization, we recommend using 5-10 ug of antibody per mg of beads.

The big difference between cells and proteins is in the detachment step. Cells and organelles need specific detachment systems, while you can elute proteins off the beads with conventional elution methods (e.g. low pH, high salt, boiling in SDS, etc.). For phage display assays, bacteriophages (bacterial viruses) are eluted by methods similar to protein elution with some modifications. The reason for this is that phages are very tolerant to different conditions, only consisting of a protein cap or wall with nucleic acids inside. Such conditions will destroy cells and organelles. Though viruses differ in some respects from phages, they also differ from cells. There is a wide range of types, e.g. some only consisting of protein cap plus nucleic acids and some with a membrane on the outside. Therefore, they can be more like phages or more like cells, depending on type. Since we have little experience with viruses, it is difficult to give straightforward suggestions as to the "best" method of choice for elution.

Another issue to consider is how much virus is present in the sample. We suggest the indirect isolation method (mix antibody and target first, immobilize complex on beads afterwards) if the target is rare.

Answer Id: E6151

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Product FAQ

Can you provide me with an overview of indirect versus direct isolation strategies using Dynabeads magnetic beads?

Answer

We would recommend using the indirect technique when:
A cocktail of mouse monoclonal antibodies is used in negative isolation to deplete several cell types simultaneously (for this, we recommend using mononuclear cells as a starting sample to remove erythrocytes, platelets, and granulocytes),
A very high depletion efficiency is required.
The mouse antibodies have low affinity.
The cells express a low number of target antigens.
The direct technique does not give satisfactory purity.

We would reccomend using the direct technique when:
The affinity of the mouse monoclonal primary antibodies is high.
The cells express a high number of target antigens.
The indirect technique is not compatible with the application of interest.
A larger stock preparation of primary antibody-coated Dynabeads magnetic beads is desired (these will generally have the same shelf life as that stated on the Dynabeads vial).

Answer Id: E6100

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Product FAQ

How can I use Dynabeads magnetic beads for triple cell isolation?

Answer

Please see the following details below for for triple cell isolation:

(1) Endothelial cell isolation:
Use Dynabeads CD31 Endothelial Cell (Cat. No. 11155D). These are paramagnetic 4.5 µm beads coated with a mouse monoclonal antibody specific for the CD31 cell surface marker (PECAM-1) expressed on all endothelial cells. The product is suitable for isolation of endothelial cells from tissue samples or reselection of cultured endothelial cells. The isolated cells are pure and ideal for culture, as this approach usually avoids contamination by fibroblasts, which would otherwise overgrow your endothelial cells. You can culture the cells with the Dynabeads magnetic beads still attached, and most of the beads will be lost after the first few passages. Very high bead concentrations should, however, be avoided as a bead-to-cell ratio of more than 10 will affect endothelial cell adherence and thereby growth. A ratio of approximately 4 beads per cell should be optimal. Good yield and purity of the isolated cells is very sample dependent. It is important to have a good single-cell suspension prior to cell isolation using the beads.

(2) Fibroblast isolation:
The fibroblasts may be isolated in a second step using an anti-Thy-1 (CD90) antibody coated onto one of our beads coated with a secondary antibody. We suggest using Dynabeads Pan Mouse IgG (Cat. No. 110-41) and coupling the Thy-1 mouse anti-human monoclonal antibody to this bead using approx. 0.1 - 1 µg antibody per 10e7 beads. Incubate for 30 min at 2 - 8 degrees C, wash off excess antibody, and resuspend the beads in PBS. The beads are then ready for use, but can be stored for several months.

(3) Smooth muscle cell isolation:
As smooth muscle cells are reported to be Thy-1 and CD31 negative, they should remain in the suspension after isolation of the endothelial cells and fibroblasts. We can't recommend a specific approach for further positive isolation of the smooth muscle cells, but the most obvious approach might be to use an antibody targeting a smooth muscle cell specific target in combination with a product such as the Dynabeads Pan Mouse IgG antibody suggested above.

Answer Id: E6140

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Manual / Product Insert

Dynabeads FlowComp Mouse CD49b for flow compatible isolation mouse NK cell

Version: 002
Catalog #
  • 11464D(Discontinued)

Product Literature

Cell concentrations in human and mouse samples - Tube-based cell isolation

Product Literature

Isolate truly untouched cells—Dynabeads negative isolation of human and mouse cells

Product FAQ

Do you have any references for glomerulus isolation using Dynabeads magnetic beads?

Answer

Takemoto M. et al. (2002) A new method for large scale isolation of kidney glomeruli from mice. Am J Pathol 161:799-805.

Takemoto M. et al. (2006) Large-scale identification of genes implicated in kidney glomerulus development and function. EMBO J 25:1160-1174.

Kiritoshi S. et al. (2003) Reactive oxygen species from mitochondria induce cyclooxygenase-2 gene expression in human mesangial cells: Potential role in diabetic nephropathy, Diabetes 52:2570-2577.

Abrahamson DR et al. (2007) Laminin compensation in collagen alpha3(IV) knockout (Alport) glomeruli contributes to permeability defects. J Am Soc Nephrol 18:2465-2472.

Dai C et al. (2006) Essential role of integrin-linked kinase in podocyte biology: Bridging the integrin and slit diaphragm signaling. J Am Soc Nephrol 17:2164-2175.

Rao VH et al. (2006) Role for macrophage metalloelastase in glomerular basement membrane damage associated with alport syndrome. Am J Pathol 169:32-46.

Cheng H et al. (2007) Overexpression of cyclooxygenase-2 predisposes to podocyte injury. J Am Soc Nephrol 18:551-559.

Akilesh S et al. (2007) Podocytes use FcRn to clear IgG from the glomerular basement membrane. PNAS 105:967-972.

We recommend following the isolation procedure described by Takemoto et al. (2002). They used inactive Dynabeads M-450 Tosylactivated (Cat. No. 140-13). You may inactivate the Dynabeads M-450 Tosylactivated beads by incubating them in an amine containing Tris buffer overnight at room temperature. The amine groups in the buffer will react with the tosyl groups on the beads and inactivate them.

Answer Id: E6153

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Product FAQ

If Thermo Fisher Scientific does not have a primary-coated Dynabeads magnetic beads product for isolating my target cell type, which alternative Dynabead magnetic beads product can I use instead?

Answer

You may use one of our secondary-coated, surface-activated, or streptavidin-coated Dynabeads magnetic beads, and coat it with a primary antibody to target your cell type.

The Dynabeads magnetic beads product you choose will depend on the primary antibody available for cell targeting and the downstream application for the isolated cells:
-For primary antibodies made in mouse, use the CELLection magnetic beads Pan Mouse IgG Kit, Dynabeads magnetic beads Goat Anti-Mouse IgG, Dynabeads magnetic beads Pan Mouse IgG, Dynabeads magnetic beads Rat Anti-Mouse IgM, Dynabeads magnetic beads Rat Anti-Mouse IgM, or Dynabeads magnetic beads Sheep-Anti Mouse IgG

-For primary antibodies made in rat, use the Dynabeads magnetic beads Sheep Anti-Rat IgG

-For primary antibodies made in rabbit, use the Dynabeads magnetic beads M-280 Sheep Anti-Rabbit IgG

-For primary antibodies made in any species, use the CELLection magnetic beads Biotin Binder Kit, Dynabeads magnetic beads Biotin Binder, Dynabeads magnetic beads FlowComp Flexi, Dynabeads magnetic beads M-450 Epoxy, or Dynabeads magnetic beads M-450 Tosylactivated

Answer Id: E4670

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Product FAQ

Can a cocktail of primary antibodies be added to a cell suspension in order to pull out several target cell populations simultaneously, using one secondary-coated Dynabeads magnetic beads product?

Answer

Yes, a cocktail of primary antibodies can be added to a cell suspension in order to pull out several target cell populations with one secondary-coated Dynabeads magnetic beads product.
The Dynabeads magnetic beads Pan Mouse IgG (110.41; 110.42) works very well with a cocktail of mouse IgGs for the simultaneous capture of multiple cell types. it is recommended that you use an indirect technique with antibody cocktails (add all Ab to cells, wash off excess Ab, then add beads to capture Ab-coated cells).

Answer Id: E4667

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Product FAQ

What should I look for when results after cell isolation using Dynabeads magnetic beads are not as expected?

Answer

We would start off by recommending you to check through the following questions.

(1)Which species are you working with, human, mouse, rat, monkey, or other? Are the antibodies specific for cells of the chosen species?

(2) Is the starting sample prepared correctly? Preparation differs for whole blood, washed blood, diluted bone marrow, DNase-treated bone marrow, mononuclear cells, buffy coat, and tissue cell suspensions.

(3) Have you used positive isolation, negative isolation, or depletion? Was the method appropriate for the target cells and downstream application? For cellular applications such as culturing, cell function assays, proliferation, flow cytometry, you generally need bead-free cells obtained by positive isolation and detachment or by negative isolation. For molecular applications such as PCR, RT-PCR, protein-lyse cells after capture on beads.

(4) What concentration of beads or ratio of beads to cells was used? For positive isolation, you need 1 x 10e7 beads per mL of sample (25 µL of beads supplied at 4 x 10e8 per mL). For depletion, you need 2 x 10e7 beads per mL sample (50 µL of beads supplied at 4 x 10e8 per mL). Aim for 4 beads per target cell.

(5) What is the sample volume and what is the concentration of cells? The average total cell concentration used is 1 - 4 x 10e7 cells per mL. Were the cells counted? This is essential for accurate recovery calculations.

(6) How were the cells and beads or cells and antibody mixed? These must be well mixed: tilted and rotated. What mixing time and temperature were used? The average is 15 - 30 minutes at 2 - 8 degrees C.

(7) Was the protocol followed exactly? Did this include the recommended buffers? Buffers should always contain protein, for example 0.1% BSA or 1% fetal calf serum.

Answer Id: E6098

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Product FAQ

Which Dynabeads magnetic beads should I use if I am interested in isolating organelles with an indirect method?

Answer

If you are not concerned about bead release you can use:
- Secondary-coated Dynabeads magnetic beads
- Dynabeads Protein A/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)

Answer Id: E12140

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Product FAQ

How do Dynabeads CELLection cell isolation kits work?

Answer

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

Answer Id: E12115

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