Isolate Untouched Mouse CD8+ T cells by Depleting Non-CD8+ T Cells

Isolate untouched mouse CD8⁺ T cells by depleting non-CD8⁺ T cells (CD4⁺ T cells, B cells, monocytes/ macrophages NK cells, dendritic cells, erythrocytes and granulocytes) from mouse spleen or lymph node cells. Other sources of mouse CD8⁺ T cells can also be used as starting material after optimization for the particular application. Isolated CD8⁺ T cells are bead- and antibody-free and are suitable for any downstream application

Principle of Isolation

Add a mixture of monoclonal antibodies against the non-CD8⁺ T cells to the starting sample. Add Mouse Depletion Dynabeads to bind to the non-CD8⁺ T cells during a short incubation. Separate the bead-bound cells by a magnet. Discard the beadbound cells and use the remaining, untouched mouse CD8⁺ T cells for any application

Description of Materials

Dynabeads are uniform, superparamagnetic polystyrene beads (4.5 μm diameter) coated with a polyclonal sheep anti-rat IgG antibody.

• 2 x 10 ml Mouse Depletion Dynabeads.  Supplied at 4 x 10⁸ beads per ml in phosphate buffered saline (PBS), pH 7.4, containing 0.1% bovine serum albumin (BSA) and 0.02% sodium azide.
  

• 2 ml Antibody Mix.  The Antibody Mix contains a mixture of rat monoclonal antibodies against mouse CD45R (B220), CD11b (Mac1), Ter-119 and CD16/32 and CD4. Supplied in PBS with 0.02% sodium azide (NaN₃).
  

• The kit will process up to 1 x 10⁹ leucocytes

Additional Materials Required

• Magnet
• 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.

Important Notes:

• BSA can be replaced by FCS.
• EDTA can be replaced by sodium citrate.
• PBS containing Ca²⁺ or Mg²⁺ is not recommended.
• Keep the buffers cold!

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

See recommended procedure to prepare cells from mouse spleen or lymph nodes.


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 leucocytes
• It is critical to follow the magnet recommendations to ensure a successful isolation.

Isolation of Mouse CD8⁺ 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 CD8⁺ T cells.

Table 1.  Volumes for mouse CD8⁺ 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 CD8⁺ 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 .