Protocols

Related Product Information

Introduction

This product is intended for positive magnetic isolation of T cells (CD90.2+ or Thy1.2+) from mouse secondary lymphoid organs such as spleen or lymph nodes. In the first step of the protocol, FlowCompTM Mouse CD90.2 Antibody is added and will bind to the target cells. In the second step T cells that have bound the specific antibodies are captured by the Dynabeads. In the third and last step, the cells are released from the Dynabeads. This kit employs an antibody specific for CD90.2 (Thy-1.2) which recognizes all peripheral T cells from Thy-1.2 expressing mouse strains Balb/c, CBA/J, C3H/He,C57BL/-, DBA, NZB/-. The CD90.2 antibody does not cross-react with CD90.1 (Thy-1.1).

Downstream Applications

Isolated cells are bead-free and may be used directly in any downstream application including flow cytometry. The cells readily proliferate in response to Dynabeads® Mouse CD3/CD28 T Cell Expander (Invitrogen Dynal Cat. no.114.52D/114.53D) as measured by incorporation of EdU (EdU Click-iT products conjugated to different fluorochromes are available from Invitrogen Molecular Probes, e.g. Cat. no. A10202).

For recommended products and protocols visit www.lifetechnologies.com/immunology.

Additional requirements

  • Isolation Buffer: Ca2+ and Mg2+ free phosphate buffered saline (PBS) (e.g.Invitrogen Gibco Cat. no.14190) supplemented with 0.1% BSA and 2mM EDTA. BSA can be replaced by human serum albumin (HSA) or 2% FBS/FCS.
  • Mixer allowing both tilting and rotation.
  • Dynal magnet: see www.lifetechnologies.com/magnets for magnet recommendations.
  • Flow cytometry antibody reagents: To observe the best baseline separation Invitrogen recommends using anti-CD3 clone 145-2C11 (Invitrogen Cat. no. MCD0304) as primary fluorescent antibody. Other clones might be blocked for optimal binding to the isolated cells. See www.lifetechnologies.com/immunology for recommended products and protocols.
  • Optional: Red blood cell lysis buffer can be used prior to the isolation procedure.
  • Optional: SYTOX® Red (Invitrogen Cat. no. S34859) is recommended for evaluation of viability.


Critical notes

  • Use a mixer that provides tilting and rotation of the tubes to ensure that Dynabeads do not settle in the tube.
  • Resuspend the Dynabeads in the vial carefully before use, i.e. vortex for >30 sec., or tilt and rotate for 5 minutes.
  • This product should not be used with Dynal MPC™-1 (Cat. no. 120.01D)
  • Avoid air bubbles during pipetting.
  • Never use less than recommended volume of Dynabeads.
  • Carefully follow the recommended pipetting volumes and incubation times.
  • Do not combine this kit with your own biotinylated antibody.
 

Protocol

Approximately 30% of mouse splenocytes and 60% of mouse lymph node cells are T cells and express CD90.2. This protocol describes isolation of highly pure CD90.2 + T cells from 5 × 10 7 mouse lymphoid cells. When working with fewer cells than 5 × 10 7, use the same volumes as indicated. When working with higher cell numbers, scale up all reagent and total volumes accordingly.

Preparations
 
Prepare a single cell suspension from mouse spleens or lymph nodes.
Resuspend the cells to 1 × 10 8 cells/ml in Isolation Buffer.
Prepare approximately 10 ml of Isolation Buffer per 5 × 10 7 cells.

Isolation procedure
  1. Use 500 μl (5 × 107 cells) from the preparation above, resuspend and add 25 μl FlowComp™ Mouse CD90.2 Antibody.

  2. Mix well and incubate for 10 minutes at 2–8°C.

  3. Add 2 ml Isolation Buffer to wash the cells, followed by centrifugation for 8 minutes at 350xg.

  4. Remove and discard the supernatant.

  5. Add 1 ml Isolation Buffer to the cell pellet and resuspend.

  6. Add 75 μl resuspended FlowComp™ Dynabeads and mix well.

  7. Incubate for 15 min at room temperature (2–8°C if required) under rolling and tilting.

  8. Place the tube on the magnet for minimum 1 minute. Carefully remove and discard the supernatant.

  9. Remove the tube from the magnet. Add at least 1 ml Isolation Buffer and resuspend the bead-bound cells by gentle pipetting 5 times.

  10. Place the tube on the magnet for minimum 1 minute. Carefully remove and discard the supernatant

  11. Remove the tube from the magnet and carefully resuspend the bead-bound cells in 1 ml FlowComp™ Release Buffer.

  12. Incubate for 10 minutes at room temperature under rolling and tilting.

  13. Mix the cells by gentle pipetting 5 times and place the tube on the magnet for 1 minute.

  14. Transfer the supernatant containing the bead-free cells to a new tube and place the tube on the magnet for 1 minute to remove all residual beads.

  15. Transfer the supernatant containing the bead-free cells to a new tube. Add 2 ml Isolation Buffer followed by centrifugation for 8 minutes at 350xg.

  16. Discard the supernatant and resuspend the cell pellet in preferred cell medium. Keep the cells on 2–8°C until further use in downstream applications.

Note: All incubations performed at room temperature can also be performed at 2–8°C, if required. To observe the best baseline separation, we recommend using anti-CD3 clone 145-2C11 (Invitrogen Cat. no. MCD0304) as primary fluorescent antibody.

Technical Advice

General troubleshooting

To avoid unspecific labeling of cells during flow staining we recommend to use 30% inactivated rat serum or Fc blocking reagents prior to staining with primary fluorescent antibody.

For better purity, repeat the washing step once or transfer the bead-bound cells to a new tube before adding the FlowComp™ Release Buffer

Isolation Buffer

PBS (phosphate buffered saline) for Invitrogen Gibco (Cat. no. 14190-094) supplemented with 0.1% BSA and 2mM EDTA. If preferred, PBS with 2% fetal calf serum (FCS) and 1mM EDTA may be used.
TOP
114.65D.indd  Rev 000     5-May-2008