USF Health

 
      


Sorting with autoMACS Separator

Prepare in advance: 

Running buffer (PBS, 2mM EDTA, 0.5% BSA) in the fridge or FACSFlow, 0.5% BSA

Rinsing buffer (PBS, 2mM EDTA) PBS, 2mM EDTA or FACSRinse

For one sorting you will need 500 ml of each. If you make the solutions they should be filtered before use (use a 0.22μm bottle top filter) to prevent clogging.

The cells should be in a single cell suspension. Pre-separation filters (such as Miltenyi 130-041-407) can be used to remove cell clumps.  For up to 108 cells, resuspend in 500ml PBS, 2mM EDTA, 0.5% BSA prior to sorting. For more cells increase the volume accordingly. Cells should have been previously labeled with the microbeads.

 

AutoMACS operation:

 

  1. Check that the black bottle has at least 200ml of 70% ethanol, and that the waste bottle is empty. Change the caps of your buffer bottles, with the autoMACS caps (with probes).  Clean the probes with ethanol before inserting in the medium.
  2. Place empty tubes under each “pos” and “neg” port, and a (15ml) tube in the sample uptake port.
  3. Turn on the autoMACS.
  4. After a while, the screen should display the main menu. You can make your selection by touching the screen. In the first run of the day it is important to run Clean. The autoMACS will run ethanol through the tubes, columns and ports, followed by rinsing solution and leaves everything in running buffer, ready for sorting. It takes ~5min.
  5. The autoMACS is now ready for sorting. Place your cell sample in any tube with a U-bottom in the uptake port. Place (5-15ml) collecting tubes under the “pos” and “neg” ports.
  6. Choose Separation from the main menu. Another menu is displayed allowing the selection of the separation program more suitable for your needs. You can select the desired program by touching the arrows on the right. When that program is highlighted you can check on the boxes on the left side of the screen the characteristics of the program, if it is the right one you can press OK and the separation will proceed automatically.
  7. The positive fraction of cells (the ones with beads attached) are recovered from the “pos1” port if a separation through a single column was selected; or from “pos2” if you decided to pass the cells through two columns (better purity, lower yield). The negative fraction (cells without beads) is always recovered from the “neg” port.
  8. When the sort is completed the main menu is displayed again. Place again the 50ml Falcon tubes in the collection ports and a 15 ml tube in the uptake port. If you have more samples to sort, you should run Rinse between the sorts. The Rinse program runs rinsing buffer through the system to remove any cells left behind. If there is another user in the booking sheet for the same day, run Clean. This program passes ethanol through all the tubes, before filling the system with running buffer. If you are the last user of the day select Sleep from the Wash menu and hit the power button in the upper right corner. This program washes everything with ethanol, and leaves all the tubes and columns filled with it. You just have to switch off the main switch when the screen prompts you to do so.

 

Do not forget to empty the “waste” bottle and to store your buffers!