Introduction

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).

Materials Supplied

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.

Ordering Information

Sku Name Size Price Qty
11441D Dynabeads® Mouse Pan B (B220) 5 mL USD 886.00

Protocols

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.

Important Notes

  • 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.

Positive
isolation 
Depletion
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

  1. Add the appropriate volume of Dynabeads to the prepared sample (for volumes see 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 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.


General Information

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

Description of Materials

Dynabeads FlowComp™ are uniform, superparamagnetic beads (2.8 μm in diameter). Supplied at a concentration of approx. 1 × 109 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 (NaN3) as pereservatives.

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. Giordia E et al. Modular usage of the HLA-DRA promoter in extra-hematopoietic and hematopoietic cell types of transgenic mice. FEBS Journal 2005; 272:3214–3226

  2. Zeytin H et al. Combination of a Poxvirus-Based Vaccine with a Cyclooxygenase-2 Inhibitor (Celecoxib) Elicits antitumor Immunity and Long-Term Survival in CEA.Tg/MIN Mice. CancerResearch 2004; 64:3668–3678.

  3. Liu et al. Sle1ab Mediates the Aberrant Activation of STAT3 and Ras-ERK Signaling Pathways in B Lymphocytes  The Journal of Immunology, 2005; 174:1630–1637.

  4. Gardby E et al. Impaired CD40 - signalling in CD19-deficient mice selectively affects T2-deperdent isotype switching. Scand. J. Immunol. 2001; 53:13-23

  5. Bai YL et al. L-selectin-dependent lymphoid occupancy is required to induce alloantigen-specific tolerance. J  Immunol. 2002; 168(4):1579-1589

  6. Hare KJ et al. Induction of thymocyte positive selection does not convey immediate resistance to negative selection. Immunology. 2002; 105(2):163-170

  7. Hardy R et al. Resolution and Characterization of Pro-B Pre-Pro-B Cell Stages in Normal Mouse Bone Marrow. J Exp. Med. 1991; 173 (5): 1213-1225.
114.41D.indd    Rev 007      5-May-2006