MACS Depletion

Last Updated 11/11/98

Materials	Qty	Location	Order Info
Strepavidin magnetic beads		Fridge 7 shelf above Gina’s in a box marked EW
RPMI, 4% FCS w/ EDTA
RPMI, 4% FCS w/o EDTA
Depletion antibodies		Fridge #5
MACS Depletion Column (Type CS)		Over Manuela’s bench in kit	Miltenyl Biotec 413-05
10mL syringe		In kit	BD 300912
3-way stopcock		In kit	Maersk Medical DK-3390
MACS depletion apparatus		In cabinet under Bacchus

Use deficient RPMI with 4% FCS & 5mM EDTA to wash cells and for all subsequent steps.

Stain 200x10⁶ cells in ~200uL using the following antibodies:

Usual depletion combination

20uL CD8 biotin (8a6)

4uL CD 14 biotin (Xb9)

40uL CD20 biotin (Xb18)

Stain for 30 minutes at RT. Then wash 2X with 4mL

Stain with avidin beads (1:5) in 200uL (40 uL beads) at RT for 20 minutes.

Wash 2X with 4mL. Resuspend in 4mL.

Setting up the column

1) Unpack the column, syringes and stopcock and assemble. Fill the syringe with RPMI media (with FCS, EDTA), and push the media through the column from the bottom to remove bubbles. Do this slowly while tapping the column to loosen bubbles.

2) Make sure the media fills the column. Then use the syringe to push the media up and down the column to further loosen air bubbles from the steel wool. Make sure that air does not touch the top of the column bed.

Run the samples through column at 3mL/min. (Use 22Gx15mm needle)

Collect ~40mL flow through in a 50mL tube

We usually get ~20 million live cells (75% viability). Spin down these cells and resuspend in 2mL of deficient RPMI with 4%FCS, no EDTA

Collect retentate for use as comps.