Standard Operating Procedure

 

MACS purification of dendritic cells

 

Hazards:

*

Protection: Wear gloves

*

Waste: Drain

*

Spill Clean-Up:

*

Procedure:

ManufacturerÕs protocol recommends use of MACS buffer (1x PBS supplemented with 0.5%BSA and 2mM EDTA), we usually use 1xPBS + 1%FCS

 

  1. Isolate single cell suspension by standard preparation method (collagenase D digestion)
  2. Pass cells through 30mm nylon mesh of filter
  3. Resuspend cells in 400ml of buffer per 108 total cells (for fewer cells use the same volume)
  4. Add 100ml of anti-CD11c MicroBeads per 108 total cells and incubate 15 min in refrigirator at 6o-12o
  5. Wash cells 2x in MACS buffer
  6. Run cells through 30mm nylon mesh of filter
  7. Choose positive selection column (MS/RS for up to 107 positive cells, LS/VS for up to 108 positive cells) or a depletion column (AS for up to 3x107 positive cells, BS for up to 108 positive cells, CS for up to 2x108 positive cells)
  8. Wash the column with appropriate amount of buffer (MS/RS: 500ml, LS/VS: 3 ml)
  9. Apply cell suspension in appropriate amount of buffer onto the column (MS/RS: 500-1000ml, LS/VS: 1-10 ml), let the negative cells pass through and rinse with appropriate amount of buffer (MS/RS: 3x500ml, LS/VS: 3x3 ml). Collect effluent as negative fraction.
  10. Remove column from magnet, place column on a suitable collection tube, pipette appropriate amount of buffer (MS/RS: 1ml, LS/VS: 5ml) onto the column and flush out positive cells using the plunger

 

Notes:

SA-MicroBeads, and anti-Fitc MicroBeads antibodies are available. Also anti-Fitc MicroBeads that can be clipped off are available (Anti-Fitc MultiSort Kit). To search for other products go to www.miltenyi.com.