Isolate or Deplete CD45+ - Whole Blood, Buffy Coat, MNC Suspensions

Isolate or deplete CD45⁺ cells directly from whole blood, buffy coat or MNC suspensions with Dynabeads CD45. For rapid and consistent results in protein or gene expression analysis, lyse the CD45⁺ cells while they are still attached to the beads and directly process for further molecular analysis. Enrich circulating tumor cells (non-hematopoietic) from MNC suspensions with Dynabeads CD45.

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).
• Depletion—discard the bead-bound cells and use the remaining, untouched cells (e.g enriched tumor cells) for any application.

Description of Materials
Dynabeads CD45 are uniform, superparamagnetic polystyrene beads (4.5 μm diameter) coated with a monoclonal mouse IgG2a antibody specific for an epitope common to all known CD45 isoforms.

Materials Supplied

• 5 ml Dynabeads CD45
• 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 ).
• This product will process up to 5 x 10⁸cells

Additional Materials Required
Magnet (Dynal MPC™)

• Mixer allowing both tilting and rotation.
• Buffer 1: PBS (without Ca²⁺ and Mg²⁺ ) w/0.1% BSA and 2 mM EDTA, pH 7.4.
• Buffer 2: RPMI 1640 w/1% fetal calf serum (FCS), 1 mM CaCl₂ and 4 mM MgCl₂, pH 7.0-7.4.
• Optional: DNase I 10,000-20,000 IU/ml.

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

MNC preparation from whole blood, buffy coat or bone marrow.

• Resuspend MNC from blood or buffy coat (PBMC) at 2 x 10⁷ cells per ml in Buffer 1 (2-8°C).
• Resuspend MNC from bone marrow (BM-MNC) at 2 x 10⁷cells per ml in Buffer 2 (18-25°C, RT).

DNase treatment of BM-MNC to remove interfering DNA.

BM-MNC should be frozen in the presence of 120 IU DNase solution per 1 ml cell suspension.

1. Add 120 IU DNase I (not included) per ml BM-MNC.
2. Incubate for 30 min at RT with gentle tilting and rotation.
3. Centrifuge at 600 x g for 10 min at RT and discard the supernatant.
4. Resuspend cells in the same volume of Buffer 1.
5. Repeat step 3 once.
6. Resuspend at 2 x 10⁷ cells per ml in Buffer 1 (2-8°C).

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 100 μl Dynabeads per ml of 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.

* If the concentration of cells is increased, the volume of Dynabeads must be increased accordingly 2 x 10⁸ cells

Enrichment of Tumor Cells

Before cell isolation or transferring the enriched cells to a new tube, pre-coat the tubes for 5 min using buffer with FCS (e.g. Buffer 2).

1. Add the appropriate volume of Dynabeads (according to table 1) to the pre-coated tube. Place in a magnet for 1 min and discard the supernatant.
2. Add the prepared sample to the beads and Incubate for 30 min at 2-8°C with gentle tilting and rotation.
3. Remove the tube from the mixer and dilute the sample 40 times with Buffer 1. Mix gently.
4. Place the tube in a magnet for 10 min.
5. Transfer the supernatant to a new pre-coated tube. Fill the tube to 50 ml with Buffer 1.
6. Centrifuge at 600 x g for 15 min at 2-8°C.
7. Resuspend the cells in an appropriate volume of Buffer 2 depending on the downstream assay.

Detection of Tumor Cells

Enriched tumor cells can be detected and analyzed by immunocytochemistry (ICC).

1. Apply 400 μl cell suspension per 16 mm cytospin spot.
2. Centrifuge cells onto poly-L-lysine coated glass slides at 1,000 rpm for 3 min.
3. Discard most of the supernatant.
4. Centrifuge slides for 1 min at 2,500 rpm.
5. Leave slides to dry at RT for 30 - 60 min (or overnight).
6. Stain with appropriate antibody conjugates (e.g. AE1/AE3 or A45-B/B3) and visualize the tumor cells with alkalic phosphatase (AP)-reaction.
7. Counter stain with heamatixylin to visualize all nucleated cells.

Other Cellular Applications

Enriched tumor cells isolated with Dynabeads CD45 are bead-free, viable and can be grown in culture.

Cell Isolation (general)

Add Dynabeads to the prepared sample according to table 1.

1. Incubate for 20 min (positive isolation) or 30 min (depletion) at 2-8°C with gentle tilting and rotation.
2. Optional: increase the volume with Buffer 1 to limit trapping of unbound cells.
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 10⁷ Dynabeads.
               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.
   

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.

TOP

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