Isolation (1-3) or depletion (4,5) of murine B cells directly from spleen or lymph node cell suspensions or other samples containing B cells. For rapid and consistent results in protein or gene expression analysis, lyse the B cells while still attached to the beads and directly process for further molecular analysis.
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 beadbound cells are separated by a magnet.
- Positive isolation – discard the supernatant and use the bead-bound cells for downstream applications.
- Depletion – discard the bead-bound cells and use the remaining, untouched cells for any application.
Description of Materials
Dynabeads Mouse pan B (B220) are uniform, superparamagnetic polystyrene beads (4.5 μm diameter) coated with a monoclonal antibody specific for the B220 antigen. B220 (also known as CD45R) is expressed on all B cells throughout development, from the early pro-B stage of B cell differentiation (6).
5 ml Dynabeads Mouse pan B (B220)
4 x 108 Dynabeads/ml in phosphate buffered saline (PBS), pH 7.4, with 0.1% bovine serum albumin (BSA) and 0.02% sodium azide (NaN3).
The product will process up to 1 x 109 leucocytes (f.ex. splenocytes).
Additional Materials Required
- Magnet: (Dynal MPC™): See www.lifetechnologies.com/magnets-selection for magnet recommendations.
- Mixer allowing both tilting and rotation.
- PBS w/0.1% BSA: PBS pH 7.4 (without Ca2+ and Mg2+) with 0.1% (w/v) BSA.
- Recommended culture media: RPMI 1640 or DMEM with 10% FCS and CO2 incubation.
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 PBS w/0.1 % BSA (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 Dynabeads to the original volume (step 2) using buffer.
2.2 Sample Preparation
Mouse cells are generally obtained from spleen, thymus or lymph node, but other sources can also be used.
Please visit www.lifetechnologies.com/cellisolation and follow our QuickLinks for recommended sample preparation procedures.
- It is critical to keep the cell suspension and buffer cold (2-8°C) during incubation and separation procedures, to prevent phagocytosis of Dynabeads.
- Bead-bound cells must be handled gently. Avoid excess pipetting.
- PBS containing Ca2+ or Mg2+ is not recommended.
- BSA can be replaced by HSA or FCS. Sodium citrate can be replaced by EDTA.
- Follow the magnet recommendations to ensure a successful isolation.
Table 1: Volume of Dynabeads added per 1 ml sample. The volumes can be scaled up as required.
|Volume of cells (up to
5 x 106 leucocytes/ml)
|1 ml|| 1 ml
|Volume of Dynabeads
per 5 x 106 leucocytes
|25 μl||50 μl
|Total no. of leucocytes
processed per product
|1 x 109||5 x 108|
2.3 Depletion or Positive Isolation of B positive Mouse Cells
- Add the appropriate volume of Dynabeads to the prepared sample (for volumes see 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 bead-bound cells 3 times by resuspending in PBS 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 further mRNA, protein or other subcellular isolations.
Description of Materials
Dynabeads FlowComp™ are uniform, superparamagnetic beads (2.8 μm in diameter). Supplied at a concentration of approx. 1 × 10 9 beads (10 mg) per ml in phosphate buffered saline (PBS), pH 7.4, containing 0.1% bovine serum albumin (BSA) and 0.02% sodium azide (NaN 3) as pereservatives.
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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