Introduction

DETACHaBEAD® Mouse CD4 is intended for release of mouse CD4+ cells from Dynabeads after positive isolation with Dynabeads® Mouse CD4 (L3T4), (available separately, Cat. no. 114.45D). The released mouse CD4+ cells are pure, viable, not activated by the release-step and have no Dynabeads® or primary antibody bound to the surface.

Principle of Release

Release of Dynabeads® from the cells using DETACHaBEAD® provides a fast and reliable method of positive isolation. The cells may be isolated from mouse spleen, thymus or lymph nodes using Dynabeads® Mouse CD4 (L3T4) (not included). DETACHaBEAD® Mouse CD4 is then added to the bead-bound cells and will release the Dynabeads® and primary antibody from the mouse CD4+ T cells. Following a short incubation period, the released Dynabeads® are removed by magnetic separation and the positively selected cells collected. The isolated cells are pure (>99%), viable (>95%) and are not activated by the isolation or release procedure.

Downstream Application

The isolated cells are pure, viable and are free from antibody bound to the surface. The isolated cells can be use  for any research applications, functional studies and flow cytometry. Cells positively isolated and released in this way have been shown to respond in a normal manner when stimulated with interleukin-2 or interferon-gamma.

Description of Materials

DETACHaBEAD® Mouse CD4 is a polyclonal antibody specifically made to out-compete the binding of mouse CD4+ cells to the Dynabeads® Mouse CD4 (L3T4) (not included). The product contains 5 ml of DETACHaBEAD®, supplied in 0.15 M phosphate buffered saline (PBS) pH 7.4. 1 ml of DETACHaBEAD® is sufficient to release cells from 2 ml Dynabeads® Mouse CD4 (L3T4).

Additional Materials Required

Materials that are not included, but are needed to perform the entire protocol:

  • Dynabeads® Mouse CD4 (L3T4), (Cat. no. 114.45D) for positive isolation of mouse CD4+ cells.
  • Magnet
  • Mixer allowing both tilting and rotation.
  • Tubes: We recommend adapting the tube size to the working volume (e.g. using 1.5 ml Eppendorf tubes for small volumes).
  • Cell culture media: RPMI 1640/1% FCS (Foetal Calf Serum).


CRITICAL NOTES

  • Never resuspend the positively isolated cells in less than 100 μl Cell culture media, even if the starting sample is less than 1 x 107 MNC or 1 ml blood.
  • The volume of DETACHaBEAD® used relates to the volume of Dynabeads® used for cell isolation. Never use less than 10 μl DETACHaBEAD® per 25 μl Dynabeads® used for positive isolation. The amount of DETACHaBEAD® used can be directly scaled up.
  • Incubation at 2-8°C will significantly reduce the number of released cells. Incubating at 37°C will not increase release efficiency.

Protocol

The amount of DETACHaBEAD® needed to obtain optimal release varies with the number of Dynabeads® used. This protocol is based on an initial positive cell isolation step from a starting sample of 1 x 107 cells using 25 μl Dynabeads®. When working with less than 25 μl Dynabeads® for cell isolation, use the volumes indicated below for release. When working with > 25 μl Dynabeads®, scale up the release protocol accordingly.

  1. Perform positive cell isolation with Dynabeads® Mouse CD4 (L3T4) as recommended (for protocol, see package insert for Dynabeads® Mouse CD4 (L3T4), (Cat. no. 114.45D). Resuspend the bead-bound cells in 100 μl Cell culture media.

  2. Add 10 μl DETACHaBEAD per 25 μl (1 x 107) Dynabeads® used to isolate the bead-bound cells in the positive isolation protocol.

  3. Incubate for 45 min at room temperature with gentle mixing.

  4. Place the tube in a magnet for 1 min.

  5. Transfer the supernatant containing released cells to a fresh tube. To obtain residual cells, wash the beads 2-3 times in 500 μl Cell culture media and collect the supernatant.

  6. Wash released cells thoroughly by resuspending the cells in a total volume of 10 ml Cell culture media and centrifuge for 6 min at 400 x g to remove DETACHaBEAD®.

  7. Resuspend the cells in Cell culture media or other media and use in downstream application.
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References

  1. Harrington LE et al (2005) Interleukin 17- producing CD4+ effector T cells develop via a l inage distinct from the T helper type 1 and 2 lineages. Nat. Immunol. 6 (11): 1123-1132.

  2. Henderson WR et al (2007) Importance of group X-secreted phospholipase A2 in allergen- induced airway inflammation and remodelling in a m ouse asthma mode. JEM 204 (4): 865-877.

  3. Maynard CL et al (2007) Regulatory T cells expressing interleukin 10 develop from Foxp3+ and Foxp3- precursor cells in the absence of interleukin 10. Nat. Immunol. 8 (9):931-941.

  4. Oeckinghouse A et al (2007) Malt1 ubiquination triggers NF-kB signaling upon T cell activation. EMBO J. 26:4634-4645.

  5. Oh-hora M et al (2007) Dual functions for the endoplasmic reticulum calcium sensors STIM1 and STIM2 in T cell activation and tolerance. Nat. Immunol. 9 (4):432- 443.

  6. Tanaka Y et al (2007) T helper type 2 differentiation and intracellular trafficking of the interleukin 4 receptor-a subunit controlled by the Rac activator Dock2. Nat. Immunol. 8 (10):1067-1075.

  7. Thakker P et al (2007) IL-23 is critical in the Induction but Not in the Effector Phase of Experimental Autoimmune Encephalomyelitis. J. Immunol. 178:2589- 2598.
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124.06D.indd  Rev 006  10-Aug-2008