4. Carefully remove and collect the upper fraction corresponding
to plasma (using a 25 mL pipette).
5. Collect PBMC by carefully pipetting the white ring located at
the interface between the plasma (top) and the Ficoll (bottom)
layer and add to the plasma fraction (using a 2 mL
suction bulb).
6. Wash PBMC two times by centrifugation at 330 Â g for 10 min
at room temperature and resuspension of the pellet in 45 mL
DPBS-FBS in 50-mL centrifuge tubes.
7. Resuspend and pool PBMC pellets in 45 mL DPBS-FBS in
50-mL centrifuge tubes.
8. Count cells using a counting chamber (expect to obtain about
6 Â 10
8 cells per Buffy coat).
9. Centrifuge at 330 Â g for 10 min at room temperature.
10. Resuspend the pellet in cold (4
C) PBMC-buffer for monocyte purification by magnetic separation or in FBS supplemented with 10% DMSO to reach a 2 Â 10
7 cells/mL for longterm storage (see Note 19).
11. Resuspend cell pellet in the appropriate volume of cold PBMCbuffer, based on the use of 80 μL per 10
7 total cells.
12. Incubate for 5 min on ice.
13. Add the appropriate quantity of CD14
+ magnetic beads, based
on the use of 10 μL per 10
7 total cells.
14. Incubate for 15 min at 4
C, using a rotating shaker to
homogenize.
15. Dilute cells in 15 mL of cold PBMC-buffer.
16. Centrifuge at 330 Â g for 10 min at 4
C.
17. Resuspend the pellet in the appropriate volume of cold PBMCbuffer, based on the use of 50 μL per 10
7 total cells.
18. Apply cell suspension onto a column attached to the magnet
and wash three times with 2.5 mL of cold PBMC-buffer.
19. Remove the column from the magnet and elute CD14
+ monocytes in a clean 15-mL tube by firmly flushing out the column
with 5 mL of cold PBMC-buffer.
20. Repeat steps 18 and 19 on a second column to increase the
purification rate.
21. Complete to 15 mL with DPBS.
22. Count CD14
+ PBMC using a counting chamber (expect to
obtain about 60 up to 120 Â 10
6 CD14
+ cells per Buffy coat to
prepare from 2 up to 4 Â 144 cm
2 petri dish).
23. Centrifuge cells at 330 Â g for 10 min at room temperature in
15-mL centrifuge tubes.
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Nathalie Deboosere et al.
to plasma (using a 25 mL pipette).
5. Collect PBMC by carefully pipetting the white ring located at
the interface between the plasma (top) and the Ficoll (bottom)
layer and add to the plasma fraction (using a 2 mL
suction bulb).
6. Wash PBMC two times by centrifugation at 330 Â g for 10 min
at room temperature and resuspension of the pellet in 45 mL
DPBS-FBS in 50-mL centrifuge tubes.
7. Resuspend and pool PBMC pellets in 45 mL DPBS-FBS in
50-mL centrifuge tubes.
8. Count cells using a counting chamber (expect to obtain about
6 Â 10
8 cells per Buffy coat).
9. Centrifuge at 330 Â g for 10 min at room temperature.
10. Resuspend the pellet in cold (4
C) PBMC-buffer for monocyte purification by magnetic separation or in FBS supplemented with 10% DMSO to reach a 2 Â 10
7 cells/mL for longterm storage (see Note 19).
11. Resuspend cell pellet in the appropriate volume of cold PBMCbuffer, based on the use of 80 μL per 10
7 total cells.
12. Incubate for 5 min on ice.
13. Add the appropriate quantity of CD14
+ magnetic beads, based
on the use of 10 μL per 10
7 total cells.
14. Incubate for 15 min at 4
C, using a rotating shaker to
homogenize.
15. Dilute cells in 15 mL of cold PBMC-buffer.
16. Centrifuge at 330 Â g for 10 min at 4
C.
17. Resuspend the pellet in the appropriate volume of cold PBMCbuffer, based on the use of 50 μL per 10
7 total cells.
18. Apply cell suspension onto a column attached to the magnet
and wash three times with 2.5 mL of cold PBMC-buffer.
19. Remove the column from the magnet and elute CD14
+ monocytes in a clean 15-mL tube by firmly flushing out the column
with 5 mL of cold PBMC-buffer.
20. Repeat steps 18 and 19 on a second column to increase the
purification rate.
21. Complete to 15 mL with DPBS.
22. Count CD14
+ PBMC using a counting chamber (expect to
obtain about 60 up to 120 Â 10
6 CD14
+ cells per Buffy coat to
prepare from 2 up to 4 Â 144 cm
2 petri dish).
23. Centrifuge cells at 330 Â g for 10 min at room temperature in
15-mL centrifuge tubes.
672
Nathalie Deboosere et al.