Related Product Information
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 flourochromes are available from Invitrogen Molecular Probes, e.g. Cat. no. A10202).
For recommended products and protocols visit www.invitrogen.com/immunology.
- 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.invitrogen.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.invitrogen.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.
- 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.
Prepare a single cell suspension from mouse spleens or lymph nodes.
Resuspend the cells to 1 × 108 cells/ml in Isolation Buffer.
Prepare approximately 10 ml of Isolation Buffer per 5 × 107 cells.
- Use 500 μl (5 × 107 cells) from the preparation above, resuspend and add 25 μl FlowComp™ Mouse CD90.2 Antibody.
- Mix well and incubate for 10 minutes at 2–8°C.
- Add 2 ml Isolation Buffer to wash the cells, followed by centrifugation for 8 minutes at 350xg.
- Remove and discard the supernatant.
- Add 1 ml Isolation Buffer to the cell pellet and resuspend.
- Add 75 μl resuspended FlowComp™ Dynabeads and mix well.
- Incubate for 15 min at room temperature (2–8°C if required) under rolling and tilting.
- Place the tube on the magnet for minimum 1 minute. Carefully remove and discard the supernatant.
- Remove the tube from the magnet. Add at least 1 ml Isolation Buffer and resuspend the bead-bound cells by gentle pipetting 5 times.
- Place the tube on the magnet for minimum 1 minute. Carefully remove and discard the supernatant
- Remove the tube from the magnet and carefully resuspend the bead-bound cells in 1 ml FlowComp™ Release Buffer.
- Incubate for 10 minutes at room temperature under rolling and tilting.
- Mix the cells by gentle pipetting 5 times and place the tube on the magnet for 1 minute.
- 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.
- 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.
- 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.
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
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.