Magnetic Isolation or Depletion of CD2+ Cells - Whole Blood, Buffy Coat, MNC Suspensions

This protocol is intended for magnetic isolation or depletion of CD2⁺cells directly from whole blood, buffy coat or MNC. For rapid and consistent results in protein or gene expression analysis, lyse the CD2⁺ cells while they are still attached to the beads and proceed with the lysate for further molecular analysis. If bead-free T cells are required, please see list of recommended products.

Principle of Isolation

Dynabeads are mixed with the 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 (e.g. protein or gene expression analysis).
• Depletion – discard the bead-bound cells and use the remaining, untouched cells for any application.

Sample Preparation

Isolate T cells directly from whole blood, buffy coat or MNC. 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

Materials that are not included, but are needed to perform the entire protocol:
• Mixer allowing both tilting and rotation
• Magnet: See Dynabeads® mRNA Purification Kit for mRNA Purification from Total RNA preps for magnet recommendations
• 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

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 at the bottom of the tube.
• Follow the recommended volumes and incubation times.
• Avoid air bubbles during pipetting.

This protocol describes magnetic labeling and isolation of CD2+ T cells from whole blood, buffy coat or MNC.

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

* When working with lower cell concentrations, use the same volumes as indicated. When working with higher cell concentrations, scale up all reagent volumes and total volumes accordingly.

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 (bullet point 2).

Depletion or Positive Isolation of CD2⁺ 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 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 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 (lysate) to a new tube for further processing.

1. Colucci S et al (2004) T cells support osteoclastogenesis in an in vitro model derived from human multiple myeloma bone disease: the role of the OPG/TRAIL interaction. Blood 104: 3722-3730.


2. Decker T et al (2003) Rapamycin-induced G1 arrest in cycling B-CLL cells is associated with reduced expression of cyclin D3, cyclin E, cyclin A and surviving. Blood 101:278-285.


3. Ma W et al (2004) Dexamethasone inhibits IL-12p40 production in lipo-polysaccharide- stimulated human monocytic cells by down-regulating the activity of c- Jun N-terminal kinase, the activation protein- 1 and NF-kB transcription factors. J. Immunol. 172:318-330.


4. Sato K et al (2003) Modified myeloid dendritic cells act as regulatory dendritic cells to induce anergic and regulatory T cells. Blood 101:3581-3589.


5. Slager EH et al (2004) Identification of multiple HLA-DR-restricted epitopes of the tumor associated antigen CAMEL by CD4+ Th1/Th2 lymphocytes. J. Immunol. 172:5095-5102.


6. Viglietta V et al (2004) Loss of functional suppression by CD4+CD25+ regulatory T cells in patients with multiple sclerosis. J.Exp.Med. 199: 971-979.