Protocols

Introduction

This product is intended for magnetic isolation or depletion of murine CD8+ T cells directly from spleen or lymph node cell suspensions. For rapid and consistent results in protein or gene expression analysis, lyse the CD8+ T cells while still attached to the beads and directly process for further molecular analysis. If bead-free T cells are required, we recommend using Dynabeads FlowComp™ Mouse CD8 (cat.no. 114.62D).

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 molecular applications.

Depletion – discard the bead-bound cells and use the remaining, untouched cells for any application.

Sample Preparation

Mouse cells are generally obtained from spleen, thymus or lymph node, but other sources can also be used. Please visit Pan Mouse IgG - Depletion or Positive Isolation of Cells from Different Species and follow our QuickLinks for recommended sample preparation procedures.


Additional Materials Required

  • Magnet: See Dynabeads® mRNA Purification Kit for mRNA Purification from Total RNA preps for magnet recommendations.
  • Mixer allowing both tilting and rotation
  • Isolation buffer (sterile): Ca2+ and Mg2+ free phosphate buffered saline (PBS) (e.g. Gibco cat. no. 10010-023) supplemented with 0.1% BSA and 2mM EDTA. Recommended culture media: RPMI 1640 (e.g. Gibco cat. no. 12633-012) or DMEM with 10% FCS/FBS and CO2 incubation.


Note:

  • BSA can be replaced by human serum albumin (HSA) or 2% FBS/FCS.
  • EDTA can be replaced by 0.6% sodium citrate.


Critical notes:

  • Use a mixer that provides tilting and rotation of the tubes to ensure that Dynabeads do not settle in the tube
  • When incubating Dynabeads and cells, the incubation temperature must be 2-8°C to reduce phagocytic activity and other metabolic processes.
  • Follow the recommended volumes and incubation times
  • Avoid air bubbles during pipetting.

Protocol

This protocol describes magnetic labeling and isolation of Mouse CD8+ T cells.

Table 1:  Volume of Dynabeads added per ml of sample.

 Positive isolationDepletion
Volume of cells (up to 1 x 107 leucocytes/ml)*1 ml1 ml
Volume of Dynabeads® per 107 leucocytes25 μl50 μl
Total no. of leucocytes processed per product
2 x 1091 x 109

* When working with fewer cells than 1x10 7, use the same volumes as indicated. When working with higher cell numbers, scale up all reagent volumes and total volumes accordingly.


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 (step 2).

Depletion or Positive Isolation of CD8+ T Cells

  1. Add the appropriate volume of 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 the 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 beadbound cells 3 times by resuspending in Isolation Buffer to the original sample volume, and separate using the magnet.

For molecular studies, lyse cells while still attached to the beads and transfer supernatant (lysate) to a new tube for further processing.

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 kit is for research use only.

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.

Avoid pipetting by mouth!

Certificate of Analysis (CoA) is available upon request. Material Safety Data Sheet (MSDS) is available at .

References

  1. Djouad F et al (2003) Immunosuppressive effect of mesenchymal stem cells favors tumor growth in allogenic animals. Blood 102: 3837-3844.

  2. Martin S et al (2004) Fas-mediated inhibition of CD4+ T cell priming results in dominance of type I CD8+ T cells in the immune response to the contact sensitizer trinitrophenyl. J. Immunol. 173: 3178-3185.

  3. Puliaev R et al (2004) Differential requirements for IFN-g in CTL maturation in acute murine graft-versus-host disease. J. Immunol. 173: 910-919

  4. Gorbachev AV et al (2004) CD40 engagement enhances antigen-presenting Langerhans cell priming of IFN-g-producing CD4+ and CD8+ T cells independently of Il-12. J. Immunol. 173: 2443-2452.


TOP
114.47D.indd  Rev 002      5-May-2007

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