Cell separation by direct method: Thoroughly suspend goat-anti Rat IgG magnetic particles by vortexing followed by end over end mixing for at least 1 hour at 4°C.
- Aliquot 10 µl of bead solution to clean microcentrifuge tube and wash 3X with 1 ml of cold 1X PBS (pH 7.5) or sterile media containing antibiotics.
- Add 5 µg of antibody to 20 µl 1X PBS and add to washed magnetic beads. Incubate at 4°C with agitation for at least 1 hour.
- Place tube in NEB Magnetic Separation Rack to pull beads to the side of the tube and decant supernatant being careful not to disturb bead pellet.
- Wash 4X as in step 2. Suspend beads in 100 µl of storage buffer appropriate for the primary antibody.
- Incubate primary antibody coated beads with heterogeneous cell suspension for 30 minutes at 4°C. Gently agitate the incubating suspension every 10 minutes. Use a magnetic bead to target cell ratio of greater than or equal to 5 magnetic beads per target cell. Incubation volume should be at least 1 ml for > 1 x 107 cells to reduce non-specific binding and clumping. Addition of 5% Fetal Bovine Serum to media and buffers may also serve to reduce non-specific binding.
- Magnetically separate beads to the side of the tube for at least 10 minutes. Save the supernatant for a negative selection or save the magnetic pellet for a positive selection.
- Cultured cells may detach from magnetic beads by incubating cells for up to 48 hours. Proteases such as chymopapain and trypsin can be used in some instances to release cells or interrupt antigen-antibody interaction.