Introduction

Isolate or deplete murine CD4+ T cells directly from spleen or lymph node cell suspensions. The Dynabeads® can be detached using DETACHaBEAD® (not supplied, available separately as Cat. no. 124.06D). Isolated cells are bead and antibody-free, phenotypically unaltered and ideal for any downstream application including flow cytometry, functional studies and expansion.

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.

  • Depletion – discard the bead-bound cells and use the remaining, untouched cells for any application.
  • Positive isolation – discard the supernatant and use the bead-bound cells for downstream applications (e.g. molecular analysis or cell culture).
  • Detachment (optional) – Positively isolated cells are gently released using DETACHaBEAD® Mouse CD4 (Cat. no. 124.06D).


Description of Materials

Dynabeads® Mouse CD4 (L3T4) are uniform, superparamagnetic polystyrene beads (4.5 μm diameter) coated with a rat monoclonal antibody. This antibody is specific for the L3T4 membrane antigen expressed on thymocytes and the T helper subpopulation of mature T cells of all common mouse strains.


Material Suppled

  • 5 ml Dynabeads® Mouse CD4 (L3T4)- 4 x 108 Dynabeads/ml in phosphate buffered saline (PBS), pH 7.4, containing 0.1% bovine serum albumin (BSA) and 0.02% sodium azide (NaN3).
  • The product will process up to 2 x 109 leucocytes


Additional Materials Required

  • DynaMag™ magnet:: See www.lifetechnologies.com/magnets for magnet recommendation.
  • Recommended culture media: RPMI 1640 or DMEM with 10% FCS and CO2 incubation.
  • Mixer allowing both tilting and rotation.
  • Isolation buffer: Ca2+ and Mg2+ free phosphate buffered saline (PBS) (e.g. Gibco Cat. no. 14190-094) supplemented with 0.1% BSA and 2mM EDTA.
  • DETACHaBEAD® Mouse CD4 – Cat. no. 124.06 (optional, to release cells from the beads after positive isolation).


Important Notes:

  • Keep the buffers cold.
  • BSA can be replaced by human serum albumin (HSA) or 2 % FBS/FCS.
  • EDTA can be replaced by 0.6 % sodium citrate.
  • PBS containing Ca2+ or Mg2+ is not recommended.


Critical notes:

  • The Dynabeads® should be washed before use (see ‘Dynabeads® Washing Procedure’)
  • Use a mixer that provides tilting and rotation of the tubes to ensure that Dynabeads® do not settle in the tube.
  • This product should not be used with the Dynal MPC™-1 (Cat. no. 120.01D).
  • Follow the recommended volumes and incubation times.
  • Never use less than 25 μl Dynabeads® per ml cell sample.
  • Avoid air bubbles during pipetting.
  • Keep the temperature at 2-8°C when incubation Dynabeads® and cells, to minimize phagocytic activity and other metabolic processes.


Ordering Information

Sku Name Size Price Qty
11445D Dynabeads® Mouse CD4 5 mL USD 886.00

Protocol

Dynabeads® Washing Procedure

  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®.


Preparations

Mouse cells are generally obtained from spleen, thymus or lymph node, but other sources can also be used. Resuspend the cells at 1 x 107 cells per ml Isolation buffer.

Isolation Procedure

The protocol presented below describes depletion and/or positive isolation of mouse CD4+ T cells.

Positive isolation Depletion
Volume of cell sample (up to 1 x 107 cells/ml) 1 ml 1 ml
Volume of Dynabeads® per 107 cells 25 μl 50 μl
Total no. of cells processed per product (5 ml) 2 x 109 1 x 109


Table 1: Volume of Dynabeads® added per 1 ml cell sample. The volumes can be scaled up as required.

This protocol is based on using 1 x 107 cells. When working with fewer cells than 1 x 107, use the same volumes as indicated. When working with higher cell numbers, scale up all reagent and total volumes accordingly.

  1. For positive isolation, add 25 μl Dynabeads® to the prepared cell sample (for depletion, use 50 μl Dynabeads®).

  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 beadbound cells 3 times by resuspending in 1 ml Isolation buffer and separate using a magnet.


For release of Dynabeads® from positively isolated cells, see DETACHaBEAD® Mouse CD4 package insert for protocol (Cat. no. 124.06D). For molecular studies, lyse cells while still attached to the beads and transfer supernatant to a new tube for further mRNA, protein or other subcellular isolations. For other recommended sample preparation procedures, visit /cellisolation.

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 .
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References

  1. Chandra AP et al (2007) Chemokine and toll-like receptor signaling in macrophage mediated islet xenograft rejection. Xenotranspl. 14:48-59.

  2. Dooms H et al (2007) Interleukin-2 enhances CD4+ T cell memory by promoting the generation of IL-7Ra-expressing cells. JEM 204 (3):547-557.

  3. Gwack Y et al (2007) Biochemical and Functional Characterization of Orai Proteins. J.Biol.Chem. 282 (22):16232-16243.

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

  5. Henderson WR et al (2007) Importance of group X-secreted phospholipase A2 in allergen-induced airway inflammation and remodeling in a mouse asthma model. JEM 204 (4):865-877.

  6. 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.

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

  8. Roan NR and Starnbach MN (2006) Antigen-Specific CD8+ T Cells Respond to Chlamydia trachomatis in the Genital Mucosa. J.Immunol. 177:7974-7979.

  9. Schillaci R et al (2006) Immunization with Murine Breast Cancer Cells Treated with Antisense Oligodeoxynucleotides to type I Insulin-Like Growth Factor Receptor Induced an Antitumoral Effect Mediated by a CD8+ Response Involving Fas/Fas Ligand Cytotoxic Pathway. J. Immunol. 176:3426-3437.

  10. Tager AM et al (2008) The lysophosphatidic acid receptor LPA1 links pulmonary fibrosis to lung injury by mediating fibroblast recruitment and vascular leak. Nat. Med. 14 (19):45-53.

  11. 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 Dock 2. Nat. Immunol. 8 (10):1067-1075.



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114.45D.indd  Rev 002      5-May-2008