Magnetic Separation

1. Place the MS+ column in to the magnet according to the manufacturer's instructions.

2. Flush the magnetic column with at least 0.5 mL of PBS buffer by applying it to the top of the column and allowing to run through. Discard the tube with the eluted PBS buffer and replace it with a new tube.

3. Load the column with 0.5 mL of the labeled cell suspension using a pipet, allow the cell suspension to run through, follow by 1.5 mL of buffer solution. The eluted cell suspension is depleted of CD56+ cells, and is designated as the negative cell fraction.

Fig. 1. Magnetic separation of NK cells characterized by CD56 cell surface marker. FACS histograms of the PBL sample labeled with anti CD56-PE primary antibody and anti PE-MACS bead secondary (A), and the negative (B), and positive (C) fractions from the magnetic separation experiment as described in the text. Note enrichment of the CD56+ cells in the positive cell fraction.

Fig. 1. Magnetic separation of NK cells characterized by CD56 cell surface marker. FACS histograms of the PBL sample labeled with anti CD56-PE primary antibody and anti PE-MACS bead secondary (A), and the negative (B), and positive (C) fractions from the magnetic separation experiment as described in the text. Note enrichment of the CD56+ cells in the positive cell fraction.

4. Remove the column from the magnet, place over a new tube, and flush the column content with 1 mL of PBS buffer using plunger supplied by the manufacturer. The eluted cell suspension is enriched in the CD56+ cells, and is designated as the positive cell fraction.

5. Optionally, repeat step 4 to increase the CD56+ cell recovery in the positive cell fraction.

6. Optionally, apply the positive cell fraction to a new MS+ column by repeating steps 1-4 to increase the purity of CD56+ cells in the positive cell fraction.

7. Set aside 1 x 106 cell aliquots from the positive and negative cell fractions for FACS analysis. Determine total cell concentration, cell suspension volume, and population viability in the separated cell fractions.

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