MACS CD14+ Separation of monocytes

Contributed by Martin Fitzpatrick, University of Birmingham, United Kingdom

Isolation of CD14+ subset of monocytes by MACS magnetic bead isolation.

• Centrifuge whole cell population at 1200rpm 10 minutes to pellet cells
• Remove supernatant and resuspend in 80ul MACS buffer per 10^7 cells
• Add 20ul CD14+ MACS beads per 10^7 cells
• Mix thoroughly and incubate for 15 minutes at 4’C
• Wash in 1-2ml buffer per 10^7 cells and then centrifuge at 1200rpm 10 minutes to pellet
• Resuspend in 500ul buffer per 10^8 cells (5ml/10^7)
• Place MACS column in magnetic field (in stand) and rinse through with 3mls MACS buffer
• Add cell suspension to column, allow to run through collecting run-off as waste
• Wash 3x 3ml with buffer, collecting run-off as waste
• Remove column to collection tube. Add 5ml buffer wash through and plunger to collect
• Count resulting cells and purity check by flow cytometry