Isolate Untouched Mouse T Cells by Depleting Non-T Cells

• 2 x 10 ml Mouse Depletion Dynabeads.

• 2 ml Antibody Mix.

• The kit will process up to 1 x 10⁹ leucocytes.

• Magnet: (Dynal MPC™) - MPC-L for 1-5 ml samples, MPC-15 for 1-15 ml samples and MPC-50 for 15-50 ml samples.
• Heat inactivated Fetal Calf Serum
• (FCS).
• Mixer allowing both tilting and rotation.
• Buffer 1: PBS (without Ca²⁺ and Mg²⁺) w/0.1% BSA and 2 mM EDTA, pH 7.4.
• Buffer 2: RPMI-1640 w/10% FCS.

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 Buffer 1, 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 Buffer 1 as the initial volume of Dynabeads.

Sample Preparation

For a recommended procedure to prepare cells from mouse spleen or lymph nodes, visit /samplepreparation.

Critical Steps for Cell Isolation

• Use a mixer that provides tilting and rotation of the tubes to ensure Dynabeads do not settle at the bottom of the tube.
• Never use less than 200 μl Dynabeads per 1 x 10⁷ MNC sample.
• It is critical to follow the magnet recommendations to ensure a successful isolation.

Isolation of Mouse T Cells fromSpleen or Lymph Node Leucocytes

This protocol is based on 1 x 10⁷ leucocytes. It is scalable from 1 x 10⁷-1 x 10⁹ cells, (see table 1).

1. Transfer 100 μl (1 x 10⁷) leucocytes in Buffer 1 to a tube.


2. Add 20 μl heat inactivated FCS.


3. Add 20 μl of Antibody Mix.


4. Mix well and incubate for 20 min at 2-8°C.


5. Wash the cells by adding 2 ml Buffer 1. Mix well by tilting the tube several times and centrifuge at 300 x g for 8 min at 2-8°C. Discard the supernatant.


6. Resuspend the cells in 800 μl Buffer 1.


7. Add 200 μl pre-washed Mouse Depletion Dynabeads.


8. Incubate for 15 min at 18-25°C with gentle tilting and rotation.


9. Resuspend the bead-bound cells by gently pipetting 5 times using a pipette with a narrow tip opening, (e.g. a 1000 μl pipette tip or a 5 ml serological pipette).


10. Add 1 ml Buffer 1.


11. Place the tube in the magnet for 2 min.


12. Transfer the supernatant to a new tube.

The supernatant contains the negatively isolated mouse T cells.

Table 1.  Volumes for mouse T cell isolation per 1 x 10⁷ starting leucocytes.

When working with higher cell numbers, scale up all reagents and volumes accordingly (e.g. for 2 x 10⁷ leucocytes use twice the volume of all indicated reagent volumes). As a rule of thumb, up to 5 x 10⁷ leucocytes can be processed in a single 15 ml tube and up to 2 x 10⁸ leucocytes can be processed in a single 50 ml tube.

Downstream Applications

Isolated mouse T cells can be used in applications such as cell culture, flow cytometry, functional assays and molecular studies.

Invitrogen Dynal AS complies with the Quality System Standards ISO 9001:2000 and ISO 13485:2003.

Description of Materials

Dynabeads FlowComp™ are uniform, superparamagnetic beads (2.8 μm in diameter). Supplied at a concentration of approx. 1 × 10⁹ beads (10 mg) per ml in phosphate buffered saline (PBS), pH 7.4, containing 0.1% bovine serum albumin (BSA) and 0.02% sodium azide (NaN₃) as preservatives.

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 product is for research use only. Not intended for any animal or human therapeutic or diagnostic use unless otherwise stated. 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. Certificate of Analysis (CoA) is available upon request. Material Safety Data Sheet (MSDS) is available at .