Search Thermo Fisher Scientific
Search Thermo Fisher Scientific
Dynabeads Biotin Binder in combination with biotinylated antibodies are ideal for depletion or positive isolation of cells from any species depending on the specificity of the antibody. Other biotinylated molecules (e.g., peptides/proteins, lectins, nucleic acids) may also be used depending on the target. Cells can be directly isolated from any sample, such as whole blood, bone marrow, mononuclear suspensions, or tissue digests.
Note: If you want to perform positive cell isolation with biotinylated antibodies followed by bead detachment, please use CELLection™ Biotin Binder (Cat. no. 161.02). If you want to use biotinylated ligands for molecular applications such as protein purifications, DNA/RNA binding protein isolations, preparing single-stranded templates, etc., please see www.lifetechnologies.com for other streptavidin-coated beads.
Principle of Isolation
Either add the biotinylated antibody to the cell sample (indirect technique) or pre-coat onto the beads (direct technique) prior to cell isolation. Mix the Dynabeads with the cell sample in a tube. The Dynabeads will bind to the target cells during a short incubation, and then separate the beadbound cells by a magnet.
Description of Materials
Dynabeads Biotin Binder are uniform, superparamagnetic polystyrene beads (2.8 μm diameter) coated with recombinant streptavidin. The streptavidin coated onto Dynabeads will bind most biotinylated ligands. Unwanted binding of cells to streptavidin via lectin-like receptors or other adhesive receptors is avoided since the recombinant streptavidin contains neither sugar nor the RYDsequence.
Materials Supplied
5 ml Dynabeads Biotin Binder 4 x 108 beads/ml in phosphate buffered saline (PBS), pH 7.4, containing 0.1% BSA and 0.02% sodium azide (NaN3). This product will process up to 2 x 109 cells.
Additional Materials Required
Important Notes:
Serum or serum albumin fractions may contain free biotin and should not be used. Dynal recommends Bovine Serum Albumin fraction V from Sigma (cat. no. A4503 or equivalent), which does not contain free biotin. Do not use culture media as it may contain free biotin. BSA can be replaced by other biotin-free blocking proteins or 1 mg/ml pluronic (F68). EDTA can be replaced by sodium citrate. PBS containing Ca2+ or Mg2+ is not recommended.
Dynabeads Washing Procedure
Dynabeads should be washed before use.
Sample Preparation
Whole Blood and Buffy Coat
Whole blood and buffy coat may be washed before use to remove interfering factors.
1. Dilute the whole blood or buffy coat in Buffer 1 (1+2).
2. Centrifuge at 600 x g for 10 min at RT (18-25°C).
3. Discard the plasma fraction/upper layer.
4. Resuspend to the original volume in Buffer 1 (2-8°C).
MNC Preparation from Whole Blood, Buffy Coat, or Bone Marrow
1. Resuspend at 1 x 107 cells per ml in Buffer 1 (2-8°C).
Please contact technical center for a list of recommended human and mouse 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 (1 x 107) 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 (1 x 107 cells/ml*) | 1 ml Max 2.5 x 106 target cells | 1 ml Max 2.5 x 106 target cells |
Volume of Dynabeads | 25 μl | 50 μl |
* If the concentration of cells is increased or the target cell concentration exceeds 2.5 x 106, the Dynabeads volume must be increased accordingly. Cell concentration can be up to 1 x 108 cells per ml.
Cell Isolation - Indirect Technique
Labeling Cells with Biotinylated Antibodies or Ligands
Label cells with biotinylated antibody as recommended by the manufacturer. If necessary, optimize incubation time, antibody titer, and cell concentration for best signal to noise ratio. Use approximately 1 μg biotinylated antibody per 106 target cells (if no other recommendation is stated) and ≥ 1 x 107 cells/ml.
Isolation or Depletion of Cells
i). Add 1 ml Buffer 1 per 1 x 107 Dynabeads.
ii).Place the tube in the magnet for 2 min and discard the supernatant.
Cell Isolation - Direct Technique
Pre-coating Dynabeads
Isolation or Depletion of Target Cells
i). Add 1 ml Buffer 1 per 1 x 107 Dynabeads.
ii).Place the tube in the magnet for 2 min and discard the supernatant.
7. Resuspend the cells in buffer/medium for downstream application.
Indirect versus Direct Technique
Use the indirect technique when:
The direct technique may be used if:
Antibody Selection
The choice of biotinylated antibody is the most important factor for successful cell isolation. Note that some antibodies may show reduced antigen-binding efficiency when coated onto beads (direct technique), even though the antibody shows good results in other immunological assays. Some antibodies may be delivered in buffers containing high levels of free biotin. To be able to precoat the Dynabeads (direct technique), the free biotin needs to be removed (e.g., dialysis or spin column with MW 10, 000 cut-off).
Labeling Cells with Biotinylated Antibodies
Isolation and Depletion of Target Cells
For Research Use Only. Not for use in diagnostic procedures.