Related Product Information
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.
- 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 www.invitrogen.com/magnets-selection 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.
Dynabeads should be washed before use.
- Resuspend the Dynabeads in the vial.
- Transfer the desired volume of Dynabeads to a tube.
- Add the same volume of Buffer 1, or at least 1 ml, and mix.
- Place the tube in a magnet for 1 min and discard the supernatant.
- Remove the tube from the magnet and resuspend the washed Dynabeads in the same volume of Buffer 1
- 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
- Dilute the whole blood or buffy coat in Buffer 2 (1+2).
- Centrifuge at 600 x g for 10 min at 18-25°C.
- Discard the plasma fraction/upper layer.
- Resuspend to the original volume in Buffer 2 (2-8°C).
Please visit www.invitrogen.com/cellisolation and follow our QuickLinks for recommended sample preparation procedures.
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.
|Sample volume - ||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
- Add the appropriate volume of Dynabeads to the prepared sample according to table 1.
- Incubate for 20 min (positive isolation) or 30 min (depletion) at 2 - 8°C with gentle tilting and rotation.
- Place the tube in a magnet for 2 min.
- For depletion, transfer supernatant to a new tube for further use.
- 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.
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 http://www.invitrogen.com.
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