Protocols

Introduction

Isolate or deplete CD19+ B cells directly from whole blood, buffy coat or MNC suspensions. For rapid and consistent results in protein or gene expression analysis, lyse the cells while they are still attached to the beads and directly process for further molecular analysis.

Note: To detach the Dynabeads from the isolated cells, use DETACHaBEAD® CD19 (not supplied).

Principle of Isolation

Dynabeads are mixed with the 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 bead-bound cells and use the remaining, untouched cells for any application.


Description of Materials

Dynabeads CD19 are uniform, superparamagnetic polystyrene beads (4.5 μm diameter) coated with a monoclonal antibody specific for the CD19 membrane antigen.

Materials Supplied

  • 5 ml Dynabeads CD19 pan B 4 x 108beads/ml in phosphate buffered saline (PBS), pH 7.4, containing 0.1% bovine serum albumin (BSA) and 0.02% sodium azide (NaN3).
  • This product can be used to process up to 5 x 109 cells.


Additional Materials Required

  • Magnet (Dynal MPC™): See www.lifetechnologies.com/magnets-selection for magnet recommendations.
  • Mixer allowing both tilting and rotation.
  • Buffer 1: PBS (without Ca2+ and Mg2+) w/0.1% BSA and 2 mM EDTA, pH 7.4.
  • Optional: DETACHaBEAD CD19 (Cat. no. 125.06D)

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 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 as the initial volume of Dynabeads.

Sample Preparation

Cells can be directly isolated from any sample such as whole blood, bone marrow, MNC suspensions or tissue digests.

Please visit www.invitrogen.com/samplepreparation for a list of recommended sample preparation procedures.

Critical Steps for Cell Isolation

  • Use a mixer that tilts and rotates 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 cell sample and at least 4 Dynabeads per target cell.

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

  Positive isolation Depletion
Sample volume
(e.g. 2.5 x 107 cells/ml)
Max 2.5 x 106 target cells/ml*
1 ml
1 ml 
Volume of Dynabeads25 μl50 μl
Total no. of cells
processed per product
5 x 109 cells
2.5 x 109cells

* If the concentration of target cells is increased, the volume of Dynabeads must be increased accordingly. Cell concentration can be up to 1 x 10 8 cells per ml.

Cell Isolation

  1. Add 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. Depletion: Transfer the supernatant containing the unbound cells to a fresh tube for further experiments.

  5. Positive isolation: Discard the supernatant and gently wash the bead-bound cells 4 times, using the following procedure:


                i) Add 1 ml Buffer 1 per 1 x 107Dynabeads.

                ii) Place the tube in the magnet for 1 min and discard the supernatant.

  6. Resuspend the cells in buffer/medium for downstream application. For molecular studies, lyse cells while still attached to the beads and transfer supernatant to a new tube for protein or gene expression analysis.


Detachment of Dynabeads from Isolated Cells

Efficient detachment of Dynabeads CD19 from the positively isolated cells is achieved by using DETACHaBEAD® CD19 (Cat. no. 125.06D) – see protocol at www.invitrogen.com for details.

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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111.43D.indd    Rev 002   5-May-2007

For Research Use Only. Not for use in diagnostic procedures.