5. Pellet cells by centrifugation at 220 Â g for 5 min at room
temperature.
6. Resuspend cells in 10 mL of cold RPMI-FBS and count the
cells using a counting chamber (expect to obtain
3–4 Â 10
7 cells per mouse).
7. Repeat step 5 and resuspend the pellet in pre-warmed (37
C)
RPMI-FBS for macrophage and dendritic cell differentiation
and culture (see Subheadings 3.8 and 3.9) or in FBS supplemented with 10% DMSO to reach a 2 Â 10
7 cells/mL for longterm storage in liquid nitrogen (see Note 5).
3.8 Culture of Murine
Bone Marrow-Derived
Macrophages (BMDM)
1. Establish macrophage monolayer by plating 1 Â 10
7 isolated
macrophage progenitor cells in 20 mL of pre-warmed (37
C)
RPMI-FBS supplemented with 40 ng/mL M-CSF (or M-CSF
containing L929 supernatant) per 75 cm
2 -tissue culture flasks,
or 2 Â 10
7 cells in 40 mL of medium per 175 cm
2 -tissue
culture flasks (see Note 5).
2. Incubate for 2–3 days at 37
C, 5% CO 2 .
3. Remove culture medium and replace with fresh pre-warmed
RPMI-FBS supplemented with M-CSF. (If confluency is
reached, follow steps 5–10 and subculture the cells into two
new 75 cm
2 or 175 cm
2 -tissue culture flasks).
4. Incubate cells for 3 days more at 37
C, 5% CO 2 .
5. Remove the medium and carefully wash the monolayer twice
using pre-warmed (37
C) DPBS.
6. Incubate cells with 3 mL (75 cm
2 flasks) or 6 mL (175 cm
2
flasks) of versene solution for 20 min at 37
C and 5% CO 2 .
7. Detach cells from the flask substrate by tapping and use a
scraper, if needed.
8. Rinse the flask with 10 mL or 20 mL of pre-warmed RPMIFBS and collect cell suspension in a sterile 50-mL
centrifuge tube.
9. Pellet cells by centrifugation at 220 Â g for 5 min at room
temperature.
10. Resuspend cells in 10 mL of pre-warmed RPMI-FBS.
11. Count cells using a counting chamber (expect to obtain
4–6 Â 10
7 cells per 175 cm
2 flask, corresponding to cell
number needed to prepare four to six 384-well plates).
12. Use for any phenotypic assay (see Subheadings 3.11–3.24).
3.9 Culture of Murine
Bone
Marrow-Dendritic
Cells (BMDC)
1. Establish dendritic cell monolayer by plating 2 Â 10
7 isolated
dendritic progenitor cells in 100 mL of pre-warmed (37
C)
RPMI-FBS supplemented with 40 ng/mL GM-CSF
(or GM-CSF containing J558 supernatant) per 500-cm
2
670
Nathalie Deboosere et al.
temperature.
6. Resuspend cells in 10 mL of cold RPMI-FBS and count the
cells using a counting chamber (expect to obtain
3–4 Â 10
7 cells per mouse).
7. Repeat step 5 and resuspend the pellet in pre-warmed (37
C)
RPMI-FBS for macrophage and dendritic cell differentiation
and culture (see Subheadings 3.8 and 3.9) or in FBS supplemented with 10% DMSO to reach a 2 Â 10
7 cells/mL for longterm storage in liquid nitrogen (see Note 5).
3.8 Culture of Murine
Bone Marrow-Derived
Macrophages (BMDM)
1. Establish macrophage monolayer by plating 1 Â 10
7 isolated
macrophage progenitor cells in 20 mL of pre-warmed (37
C)
RPMI-FBS supplemented with 40 ng/mL M-CSF (or M-CSF
containing L929 supernatant) per 75 cm
2 -tissue culture flasks,
or 2 Â 10
7 cells in 40 mL of medium per 175 cm
2 -tissue
culture flasks (see Note 5).
2. Incubate for 2–3 days at 37
C, 5% CO 2 .
3. Remove culture medium and replace with fresh pre-warmed
RPMI-FBS supplemented with M-CSF. (If confluency is
reached, follow steps 5–10 and subculture the cells into two
new 75 cm
2 or 175 cm
2 -tissue culture flasks).
4. Incubate cells for 3 days more at 37
C, 5% CO 2 .
5. Remove the medium and carefully wash the monolayer twice
using pre-warmed (37
C) DPBS.
6. Incubate cells with 3 mL (75 cm
2 flasks) or 6 mL (175 cm
2
flasks) of versene solution for 20 min at 37
C and 5% CO 2 .
7. Detach cells from the flask substrate by tapping and use a
scraper, if needed.
8. Rinse the flask with 10 mL or 20 mL of pre-warmed RPMIFBS and collect cell suspension in a sterile 50-mL
centrifuge tube.
9. Pellet cells by centrifugation at 220 Â g for 5 min at room
temperature.
10. Resuspend cells in 10 mL of pre-warmed RPMI-FBS.
11. Count cells using a counting chamber (expect to obtain
4–6 Â 10
7 cells per 175 cm
2 flask, corresponding to cell
number needed to prepare four to six 384-well plates).
12. Use for any phenotypic assay (see Subheadings 3.11–3.24).
3.9 Culture of Murine
Bone
Marrow-Dendritic
Cells (BMDC)
1. Establish dendritic cell monolayer by plating 2 Â 10
7 isolated
dendritic progenitor cells in 100 mL of pre-warmed (37
C)
RPMI-FBS supplemented with 40 ng/mL GM-CSF
(or GM-CSF containing J558 supernatant) per 500-cm
2
670
Nathalie Deboosere et al.