Introduction

Isolate or deplete CD14+ cells directly from whole blood, buffy coat, MNC suspensions or tissue digests with Dynabeads CD14. For rapid and consistent results in protein or gene expression analysis, lyse the CD14+ cells while they are still attached to the beads and directly process for further molecular analysis.

Note:
For isolation of untouched monocytes, please use the Dynabeads® Untouched™ Human Monocytes, cat no. 113.50D

Principle of Isolation

Dynabeads are mixed with the cell sample in a tube. The Dynabeads will bind to the target cells during a short incubation, and then the bead-bound cells are separated by a magnet.

  • Positive isolation – discard the supernatant and use the bead-bound cells for downstream applications (e.g. molecular analysis or cell culture).
  • Depletion – discard the beadbound cells and use the remaining, untouched cells for any application.


Description of Materials

Dynabeads CD14 are uniform, superparamagnetic polystyrene beads (4.5 μm diameter) coated with a monoclonal mouse IgG2a antibody recognising the 55kDa CD14 single-chain cell surface glycoprotein. The CD14 antigen is predominantly expressed by monocytes and macrophages.

Materials Supplied

  • 5 ml Dynabeads CD14 4 x 108 beads/ml in phosphate buffered saline (PBS), pH 7.4, containing 0.1% bovine serum albumin (BSA) and 0.02% sodium azide (NaN3 ).
  • The product will process up to 2 x 109 cells


Additional Materials Required

Materials that are not included, but are needed to perform the entire protocol:

Magnet (Dynal MPC™): See Dynabeads® mRNA Purification Kit for mRNA Purification from Total RNA preps for magnet recommendations.

  • Mixer allowing both tilting and rotation.
  • Buffer 1: PBS w/0.1% BSA, pH 7.4.
  • Buffer 2: PBS (without Ca2+ or Mg2+ ) w/0.1 % BSA and 0.6% Na-citrate or 2 mM EDTA.

Protocol

Dynabeads Washing Procedure

Dynabeads should be washed before use.

  1. Resuspend the Dynabeads in the vial.

  2. Transfer the desired volume of Dynabeads to a tube.

  3. Add the same volume of Buffer 1, or at least 1 ml, and mix.

  4. Place the tube in a magnet for 1 min and discard the supernatant.

  5. Remove the tube from the magnet and resuspend the washed Dynabeads in the same volume of Buffer 1

  6. as the initial volume of Dynabeads (step 2).


Sample Preparation - Whole Blood and Buffy Coat

Whole blood and buffy coat must be washed before use to remove soluble
CD14 (1).

  1. Dilute the whole blood or buffy coat in Buffer 2 (1+2).

  2. Centrifuge at 600 x g for 10 min at 18-25°C.

  3. Discard the plasma fraction/upper layer.

  4. Resuspend to the original volume in Buffer 2 (2-8°C).


 Please visit Pan Mouse IgG - Depletion or Positive Isolation of Cells from Different Species and follow our QuickLinks for recommended sample preparation procedures.

Cell Isolation

Critical Steps for Cell Isolation

  • Use a mixer that provides tilting and rotation of the tubes to ensure Dynabeads do not settle at the bottom of the tube. When incubating Dynabeads and cells, the incubation temperature must be 2-8°C to reduce phagocytic activity and other metabolic processes.
  • Never use less than 25 μl Dynabeads per ml of cell sample.
  • Cell concentration can be up to 1 x 108 cells per ml.


Table 1:
Volume of Dynabeads added per ml of cell sample. The volumes can be scaled up as required.

Positive isolation Depletion
Sample volume -
  • 1 x 107 MNC*/ml
  • diluted whole blood
  • diluted buffy coat
1 ml
1 ml
Volume of Dynabeads 25 μl 50 μl
Total no. of cells
processed per product
2 x 109 cells
1 x 109 cells


* if the concentration of MNC is increased, the volume of Dynabeads must be increased accordingly.

Depletion or Positive Isolation of Monocytes

  1. Add the appropriate volume of Dynabeads to the prepared sample according to table 1.

  2. Incubate for 20 min (positive isolation) or 30 min (depletion) at 2 - 8°C with gentle tilting and rotation.

  3. Place the tube in a magnet for 2 min.

  4. For depletion, transfer supernatant to a new tube for further use.

  5. For positive isolation, discard the supernatant and wash the beadbound cells 3 times by resuspending in Buffer 1 to the original sample volume, and separate using a magnet.


For molecular studies, lyse cells while still attached to the beads and transfer supernatant to a new tube for protein or gene expression analysis.

General Information

Invitrogen Dynal AS complies with the Quality System Standards ISO 9001:2000 and ISO 13485:2003.

Storage/Stability

This product is stable until the expiry date stated on the label when stored unopened at 2-8°C. Store opened vials at 2-8°C and avoid bacterial contamination. Keep Dynabeads in liquid suspension during storage and all handling steps, as drying will result in reduced performance. Resuspend well before use.

Warnings And Limitations

This product is for research use only. Not intended for any animal or human therapeutic or diagnostic use unless otherwise stated. Follow appropriate laboratory guidelines. This product contains 0.02% sodium azide as a preservative, which is cytotoxic.

Avoid pipetting by mouth!

Sodium azide may react with lead and copper plumbing to form highly explosive metal azides. When disposing through plumbing drains, flush with large volumes of water to prevent azide build up. Certificate of Analysis (CoA) is available upon request. Material Safety Data Sheet (MSDS) is available at .
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References

  1. Robinson PJ. (1991) Phosphatidylinositol membrane anchors and T-cell activation. Immunology Today 12: 35-41.

  2. Abrahamsen H et al (2004) TCRand CD28-mediated recruitment of phosphodiesterase 4 to lipid rafts potentiates TCR signaling. 173: 4847-4858.

  3. Hall A et al (2004) Rh autoantigen presentation to helper T cells in chronic lymphocytic leukemia by malignant B cells. Blood 10.1182/ blood-2003-10-3563 (pre-published online July 29, 2004).

  4. Parker B et al (2004) Alterations in vascular gene expression in invasive breast carcinoma. Cancer Research 64: 7857-7866.

  5. Decker T et al (2003) Rapamycininduced G1 arrest in cycling B-CLL cells is associated with reduced expression of cyclin D3, cyclin E, cyclin A and survivin. Blood 101: 278-285.

  6. Donaghy H et al (2003) Dysfunction and infection of freshly isolated blood myeloid and plasmacytoid dendritic cells in patients infected with HIV-1. Blood 101: 4505-4511.

  7. Holm A et al (2003) Impaired secretion of IL-10 by T cells from patients with common variable immunodeficiency-involvement of protein kinase A type I. J. Immunol. 170: 5772-5777.

  8. Ertesvag A et al (2002) Retinoic acid stimulates the cell cycle machinery in normal T cells: involvement of retinoic acid receptor-mediated IL-2 secretion. J. Immunol. 169: 5555-5563.

  9. Hirth A et al (2002) Cytokine mRNA and protein expression in primaryculture and repeated-passage synovial fibroblasts from patients with rheumatoid arthritis. Arthritis Res. 4(2): 117-125.

  10. Suarez A et al (2002) Generation of CD4+CD45RA+ Effector T Cells by Stimulation in the Presence of Cyclic Adenosine 5’-Monophosphate-Elevating Agents. J. Immunol. 1159-1167.
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111.49D.indd   Rev 002    May-5-2007