Standard Operating Procedure

 

MACS purification of CD8 T cells

 

Hazards:

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Protection: Wear gloves

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Waste: Drain

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Spill Clean-Up:

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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
  2. Pass cells through 30mm nylon mesh of filter
  3. Stain cells with FITC-conjugated antibody at appropriate time and titer (For isolation of CD44lo CD8 T cells resuspend 108 cells in 500ml of MACS buffer and antibody as determined by the following formula:

For CD4, B220 and I-Ab: number of cells in 106 x 0.125 divided by 4.5

For CD44                     number of cells in 106 x 0.04 divided by 4.5)

  1. Incubate for 20 min on ice
  2. Wash cells 2x in MACS buffer
  3. Resuspend cells in 90ml of buffer per 107 total cells
  4. Add 10ml of MACS anti-Fitc MicroBeads per 107 total cells and incubate 15 min in refrigirator at 6o-12o
  5. Run cells through 30mm nylon mesh or filter
  6. 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)
  7. Wash the column with appropriate amount of buffer (MS/RS: 500ml, LS/VS: 3 ml)
  8. 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.
  9. 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 MicroBeads conjugated directly to antibodies (i.e. CD11c) 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.