Protocols

Introduction

Isolate or deplete murine T cells directly from spleen or lymph node cell suspensions. For rapid and consistent results in protein or gene expression analysis, lyse the T cells while still attached to the beads and proceed directly to 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 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)

Materials Supplied

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.

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 Isolation Buffer, 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 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.

Important Notes

  • 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.

Positive
isolation 		Depletion
Sample volume 1 ml 1 ml
(up to 1 x 107cells/ml)		 1 ml 1 ml
Volume of Dynabeads
25 μl50 μl
Total no. of cells
processed per product		2 x 1091 x 109



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 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.


General Information

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

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.

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 .

References

  1. 1. Xu Z et al (2004) Mechanisms of peritoneal B-1A cells accumulation induced by murine lupus susceptibility locus Sle2. J. Immunol. 173:6050-6058.

  2. 2. Yazawa N et al (2003) CD19 regulates innate immunity by the toll-like receptor RP105 signaling in B lymphocytes. Blood 102:1374-1380.

  3. 3. Poe J et al (2004) Severely impaired B lymphocyte proliferation, survival and induction of the c-Myc: Cullin 1 ubiquitin ligase pathway resulting from CD22 deficiency on the C57BL/5 genetic background. J. Immunol. 172: 2100-2110.

  4. 4. Metwali A et al (2004) Cutting Edge: Hemokinin has Substance P-like function and expression in inflammation. J.Immunol. 172:6528-6532.
114.43D.indd   Rev 002      2-Dec-2008

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