• PBMC purified by Ficoll-Hypaque gradient (Protocol 8)
• RPMI1640 medium supplemented with 15% FBS
• Iso-osmotic Percoll. To prepare an osmotically balanced stock solution of
Percoll (Pharmacia) mix 9.25 parts of concentrated Percoll with 0.75 parts (v/v) of 10 x Ca2+/Mg2+-free PBS. Adjust the osmolality of the Percoll solution, as well as of the RPMI 1640 medium supplemented with 15% FBS, to 285 mOs with 10 x Ca2+/Mg2H -free PBS or distilled water, respectively.
1 Prepare a 46% Percoll solution by mixing 4.6 parts of the iso-osmotic Percoll solution with 5.4 parts of iso-osmotic complete medium.
2 Resuspend 5-10 x 107 PBMC in 5 ml of iso-osmotic complete medium.
3 Place in 10 ml centrifuge tube.
4 Slowly layer 5 ml of 46% Percoll solution underneath PBMC layer.
5 Centrifuge for 30 min at room temperature at 500 g with gradual acceleration and no brake. The monocyte-enriched fraction is collected at the interface, whereas lymphocytes are at the bottom of the tube.
Protocol 9 continued
6 Carefully remove the supernatant fluid using a pipette and discard this layer.
7 Recover the monocyte layer using a fresh pipette (avoid the Percoll layer) and transfer cells to a 50 ml conical tube.
8 Bring the total volume to 50 ml by adding Ca2+/Mg2*-free PBS.
9 Centrifuge for 5 min at room temperature at 280 g with low brake.
10 Resuspend the cell pellet in 50 ml Ca2+/Mg21 -free PBS and repeat step 9.
11 Resuspend the cell pellet in RPMI1640 medium containing 15% FBS.
12 Count the viable cells by trypan blue exclusion.
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