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Magnetic activated cell sorting (MACS) is a common method used to isolate target cells. In MACS, magnetic microparticles are coated with antibodies to cell surface markers and cells expressing this marker then become associated with the beads. The cells are then transferred to a column and a magnetic field is then applied, then when the column is washed, unwanted cells are washed away, retaining the cells of interest. MACS was instrumental in our understanding behaviour of the isolated cells in vitro.

3.2.8.1 Labelling antibody with para magnetic beads

Pierce N-hydroxysuccinimide (NHS)-Activated Magnetic Beads were used for the immunoprecipitation of the Thy-1 target cells. This required binding a specific antibody with the magnetic beads.

The binding procedure was conducted using 300μL of magnetic beads (10mg/ml) in a 1.5ml microcentrifuge tube to bind 500μL of protein solution following the manufacture’s protocol.

The coupling and blocking procedure began by equilibrating the Thy-1 antibody solution and magnetic beads to room temperature and chilling the wash buffer (PBS) on ice.

The magnetic beads were mixed by gentle tapping and repeated inversion until no clumps were seen. Each 300μL of the magnetic beads were prepared individually and were then placed into a 1.5 microcentrifuge tube in a magnetic stand (Promega). The magnetic beads were attached to the side of the tube closest to magnet in a few seconds leaving a coluorless solution in the bottom of the tube, which was discarded while the tube was inside the magnetic stand. Then the tube was removed from the magnetic stand and the beads were washed with 1ml of ice-cold PBS and gently mixed by tapping for 15 seconds until the solution was homogeneous. The tube was returned into the magnetic stand, the beads again moved toward the side wall of the tube and quickly attached to the magnetic side, leaving a colourless solution which was discarded. The tube was taken out of the magnetic stand, the Thy-1 antibody solution (500μL containing 235μg of the Thy-1 antibody) was added immediately to the tube and mixed gently by tapping for 30 seconds.

It was then incubated overnight at 4°C on a rotator (the 1.5ml microcentrifuge tube was placed in a 20ml tube and put in a horizontal rotator). During the first 30 minutes of the incubation, the solution was mixed for 10 seconds every 5 minutes. Then the tube was mixed twice for 15 seconds every 15 minutes. Then left on the horizontal rotator until incubation was complete. The bead-antibody complexes were collected with the magnetic stand and washed twice with PBS (1ml) each as previously described. One ml of ultrapure water was added and mixed for 15 seconds. The tube was placed into a magnetic stand where the bead-antibody complexes were collected and the supernatant was discarded. Then the beads were quenched by mixing with 1ml of quenching buffer for 30 seconds. The quenching buffer was 300μl of 3M ethanolamine in 5ml

of 1M HCl, pH 8.5. The pH was measured and drops of ethanolamine were added if it was acidic, but if it was alkaline HCl was added until the pH reached 8.5.

Then the tube was incubated for 2 hours at room temperature on a rotator. The beads-antibody complex was collected using the magnetic stand and the supernatant was discarded. Then 1 ml of ultrapure water was added to the bead-antibody complexes and mixed for 15 seconds. The tube was placed onto the magnetic stand, the bead-antibody complexes were again collected and the supernatant discarded. Finally, 1 ml of storage buffer [Coupling Buffer (50mM sodium bicarbonate) with 0.05% sodium azide] was added to the bead-antibody complexes and mixed for 15 seconds. The tube was placed onto the magnetic stand, the bead-antibody complexes were collected again and the supernatant was discarded. This wash was repeated two additional times and then the beads were stored in 300μL of storage buffer at 4°C until ready for use.

3.2.8.2 Binding the bead-antibody complexes to the target cells

MACS was applied to the primary adrenal cells after one day of culture in order to isolate the cells expressing Thy-1 protein in their cell membrane. The cultured cells were counted using a haemocytometer, then pelleted at 600g for 5 minutes at 4°C to remove the culture medium. Then 2 ml of sterile ice-cold PBS, containing 0.1% w/v bovine serum albumin (BSA) buffer, was used to re-suspend 2.5 million cells.

An appropriate volume (50µl) of Thy-1 antibody conjugated beads was prepared for each adrenal gland. Thy-1 antibody conjugated beads were pipetted into a 1.5 microcentrifuge tube. Then, 1 ml of PBS was added to the conjugated beads and mixed for 15 seconds. The tube was placed into a magnetic stand, the beads-antibody complexes were collected and the supernatant was discarded. This wash with PBS was repeated twice. The next step, pipetting, was conducted

slowly and gently to minimise harm to the cells. The cell suspension was added to the conjugated beads and incubated at 4°C on a rotator (Fisherbrand) with a slow rotation (20 RPM) for 30 minutes to bind the conjugated beads to the target cells. Again, the tube was placed onto the magnetic stand and the negative cells were collected. Then the tube was moved out of the magnetic stand and 1 mL of ice-cold PBS, containing 0.1% BSA, was added and gently mixed to remove any negative cells. The tube was placed onto the magnetic stand; the conjugated bead- cell complexes started to move toward the side wall of the tube and attached to the magnetic side in a few seconds leaving a clear solution, which was discarded. This wash step was repeated twice to remove negative cells.

The conjugated bead-cell complexes in each tube were re-suspended in a pre-warmed DMEM culture at 37°C and moved to one well of a 12 well plate. The plate was incubated at 37°C in a 5% CO2 humidified incubator for 10-14 days before changing the media.