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 T (Thy 1.2) are uniform, superparamagnetic polystyrene beads (4.5 μm diameter) coated with a monoclonal antibody. This monoclonal antibody is specific for the Thy 1.2 antigen expressed on peripheral mouse T cells, thymocytes and intraepithelial T lymphocytes of all common mouse strains. 5 ml Dynabeads Mouse pan B (B220)
5 ml Dynabeads Mouse pan T (Thy 1.2)
4 x 10 8 Dynabeads/ml in phosphate buffered saline (PBS), pH 7.4, with 0.1% bovine serum albumin (BSA) and 0.02% sodium azide (NaN 3).
The product will process up to 1 x 109 leucocytes (f.ex. splenocytes).
Additional Materials Required
- Magnet: We recommend DynaMag™- 15 (Prod. No. 123.01D) for 1-15 ml samples and DynaMag™-50 (Prod. No. 123.02D) for 5-50 ml samples. Dynal MPC™ magnets may also be used, except Dynal MPC-1 (Prod. No. 120.01D).
- Mixer allowing both tilting and rotation.
- Isolation Buffer: PBS pH 7,4 from Gibco (Cat.no. 10010-15 or 10010-023), supplemented with 0,1% BSA.
- Recommended culture media: RPMI 1640 from Gibco or DMEM with 10%
FCS and CO2 incubation.
Dynabeads Washing Procedure
Dynabeads should be washed before use.
- Resuspend the Dynabeads in the vial.
- Transfer the desired volume of Dynabeads to a tube.
- Add the same volume of Isolation Buffer, or at least 1 ml, and mix.
- Place the tube in a magnet for 1 min and discard the supernatant.
- Remove the tube from the magnet and resuspend the washed Dynabeads in the same volume of Isolation Buffer as the initial volume of Dynabeads.
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.
- Use a mixer that provides tilting and rotation of the tubes to ensure that the Dynabeads do not settle at the bottom of the tube.
- Keep the temperature at 2-8°C when incubating Dynabeads and cells, to minimize phagocytic activity and other metabolic processes.
- Follow the recommended volumes and incubation times.
- Never use less than 25 μl Dynabeads per ml cell sample.
- Avoid air bubbles during pipetting.
Table 1: Volume of Dynabeads added per 1 ml sample. The volumes can be scaled up as required.
|Sample volume 1 ml 1 ml
(up to 1 x 107cells/ml)
|1 ml|| 1 ml
|Volume of Dynabeads
||25 μl||50 μl
|Total no. of cells
processed per product
|2 x 109||1 x 109|
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 Isolation Buffer 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 isolation of nucleic acids, protein or other subcellular isolations.
Description of Materials
Dynabeads Mouse pan T (Thy 1.2) are uniform, superparamagnetic polystyrene beads (4.5 μm diameter) coated with a monoclonal antibody. This monoclonal antibody is specific for the Thy 1.2 antigen expressed on peripheral mouse T cells, thymocytes and intraepithelial T lymphocytes of all common mouse strains.
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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