Mouse CD4 (L3T4)

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.

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.

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.