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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Product FAQ

Do you have any references for isolation of vesicles and organelles using Dynabeads magnetic beads?

Answer

Here is a sampling of references for organelle isolation, including vesicles and exosomes.

Dynabeads products used for exosome isolation via an antibody plus some other references where Dynabeads M-500 Subcellular has been used to isolate other types of organelles:

Rabesandratana H et al. (1998) Decay-accelerating factor (CD55) and membrane inhibitor of reactive lysis (CD59) are released within exosomes during in vitro maturation of reticulocytes. Blood 91:2573-2580.

Allan BB et al. (2001)Rab1 recruitment of p115 into a cis-SNARE complex: programming budding COPII vesicles for fusion. Science 289:444-448.

Li X et al. (2001) Na+-H+ exchanger 3 (NHE3) is present in lipid rafts in the rabbit ileal brush border: a role for rafts in trafficking and rapid stimulation of NHE3. J Physiol 537:537-552.

Some references for isolation of endosomes and lysosomes.

Gagescu R (2000) The recycling of endosome of Madin-Darby canine kidney cells is a mildly acidic compartment rich in raft components. Mol Biol Cell 11:2775-2791.

Provoda CJ et al. (2000) Evidence for a primary endocytic vesicle involved in synaptic vesicle biogenesis. J Biol Chem 275:7004-7012.

Tjelle TE et al. (1998) Degradation of phagosomal components in late endocytic organelles. J Cell Sci 111:141-148.

Vitale N et al. (1998) Localization of ADP-ribosylation factor domain protein (ARD1) in lysosomes and Golgi apparatus. Proc Natl Acad Sci USA 95:8613-8618.

Clayton A et al. (2001) Analysis of antigen presenting cell derived exosomes, based on immuno-magnetic isolation and flow cytometry. J Immunol Methods 247:163-174.

Marchand S et al. (2001) Differential targeting of components of the dystrophin complex to the postsynaptic membrane. Eur J Neurosci 13: 221-229.

Some references to articles where vesicles transporting from the ER have been isolated and related references:

Allan BB et al. (2000) Rab1 recruitment of p115 into a cis-SNARE complex: programming budding COPII vesicles for fusion. Science 289:444-448.

Seemann J et al. (2002) Partitioning of the matrix fraction of the Golgi apparatus during mitosis in animal cells. Science 295:848-851.

Jesch SA, Linstedt AD (19980 The Golgi and endoplasmic reticulum remain independent during mitosis and HeLa cells. Mol Biol Cell 9:623-635.

Sinai AP et al. (1997) Association of host cell endoplasmic reticulum and mitochondria with the Toxoplasma gondii parasitophorous vacuole membrane: a high affinity interaction. J Cell Sci 110:2117-2128.

Sans N et al. (2001) Synapse-associated protein 97 selectively associates with a subset of AMPA receptors early in their biosynthetic pathway. J Neurosci 21:7506-7516.

Abo-Hashema KAH et al. (1999) Evidence for triacylglycerol synthesis in the lumen of microsomes via a lipolysis-esterification pathway involving carnitine acyltransferases. J Biol Chem 274:35577-35582.

References where Dynabeads magnetic beads have been used to isolate mitochondria and other organelles, or to isolate mitochondrial or chloroplast DNA using Dynabeads magnetic beads:

Lüers GH et al. (1998) Immuno-isolation of highly purified peroxisomes using magnetic beads and continuous immunomagnetic sorting. Electrophoresis 19:1205-1210.

Sexton PS, Cenedella RJ. (2002) Immunomagnetic capture of lens membrane fractions containing steroid binding protein. Biochem Biophys Res Commun 295:1027-1031.

Hansen GH et al. (2001) Lipid rafts exist as stable cholesterol-independent microdomains in the brush border membrane of enterocytes. J Biol Chem 276:32338-32344.

Sinai AP et al. (1997) Association of host cell endoplasmic reticulum and mitochondria with the Toxoplasma gondii parasitophorous vacuole membrane: a high affinity interaction. J Cell Sci 110:2117-2128.

Jones SM et al. (1998) Immunoisolation of organelles using magnetic solid supports, p. 12-14 in Cell Biology: A Laboratory Handbook, 2nd edn., Academic Press.

Müsch A et al. (1997) Myosin II is involved in the production of constitutive transport vesicles from the TGN. J Cell Biol 138:291-306.

Herrnstadt C et al. (1999) A novel mitochondrial DNA-like sequence in the human nuclear genome. Genomics 60:67-77.

Kausch AP et al. (1999) Organelle isolation by magnetic immunoabsorption. BioTechniques 26 (2):336-343.

Hopwood AJ et al. (1996) DNA typing from human faeces. Int J Legal Med 108:237-243.

Marchington DR et al. (1997) Homopolymeric tract heteroplasmy in mtDNA from tissues and single oocytes: support for a genetic bottleneck. Am J Hum Genet 60:408-416.

Piercy R et al. (1993) The application of mitochondrial DNA typing to the study of white Caucasian genetic identification. Int J Legal Med 106:85-90.

Fangan BM et al. (1994) A general approach for PCR-amplification and sequencing of chloroplast DNA from crude vascular plant and algal tissue. BioTechniques 16:484-494.

Ferris C et al. (1995) Using chloroplast DNA to trace postglacial migration routes of oaks into Britain. Mol Ecol 4:731-738, 1995.

Answer Id: E6150

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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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Manual / Product Insert

Exosome – Human CD81 Isolation (from cell culture)

Version: Rev. A (6 June 2014)
Catalog #

Citations & References

Isolation of cell surface-specific human monoclonal antibodies using phage display and magnetically-activated cell sorting: applications in immunohematology.

  • Authors: Siegel DL, Chang TY, Russell SL, Bunya VY
  • Journal: J Immunol Methods (1997) 206:73-85
  • PubMed ID: 9328570

Manual / Product Insert

Exosome – Human EpCAM Isolation (from cell culture)

Version: Rev. A (6 June 2014)
Catalog #

Product Literature

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

Citations & References

Human capillary endothelial cells from abdominal wall adipose tissue: isolation using an anti-pecam antibody.

  • Authors: Springhorn JP, Madri JA, Squinto SP
  • Journal: In Vitro Cell Dev Biol Anim (1995) 31:473-481
  • PubMed ID: 8589892

Manual / Product Insert

Exosome – Human CD9 Isolation (from cell culture)

Version: Rev. A (6 June 2014)
Catalog #

Citations & References

Isolation and characterization of microvessel endothelial cells from human mammary adipose tissue.

  • Authors: Hewett PW, Murray JC, Price EA, Watts ME, Woodcock M,
  • Journal: In Vitro Cell Dev Biol Anim (1993) 29A:325-331
  • PubMed ID: 7686548
Catalog #

Product Literature

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

Product Literature

Magnetic Bead Based Soil DNA Isolation using the KingFisher Flex and KingFisher Duo and ClearMag Technology

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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Citations & References

Normal human kidney HLA-DR-expressing renal microvascular endothelial cells: characterization, isolation, and regulation of MHC class II expression.

  • Authors: Muczynski KA, Ekle DM, Coder DM, Anderson SK
  • Journal: J Am Soc Nephrol (2003) 14:1336-1348
  • PubMed ID: 12707403

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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