3. Remove the medium and carefully wash the monolayer twice
using pre-warmed (37
C) DPBS.
4. 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 .
5. Detach cells from the flask substrate by tapping and use a
scraper, if needed.
6. Rinse the flask with 10 mL or 20 mL of pre-warmed RPMIFBS and collect cell suspension in a sterile 50-mL
centrifuge tube.
7. Pellet cells by centrifugation at 220 Â g for 5 min at room
temperature.
8. Resuspend cells in 10 mL of pre-warmed RPMI-FBS.
9. Count cells using a counting chamber (expect to obtain
10–20 Â 10
6 cells/mL per 75 cm
2 flask).
10. Use cells for any phenotypic assay (see Subheadings 3.11–
3.24).
11. Repeat steps 1–9 to maintain and passage A549 cells twice a
week, for a maximum of ten passages, or pellet and dilute cells
in FBS supplemented with 10% DMSO to reach a 5 Â 10
6
cells/mL suspension for long-term storage in liquid nitrogen
(see Note 3).
3.5 Production
of L929-Derived M-CSF
Conditioned Medium
for Murine Primary
Macrophage
Differentiation
and Culture [17]
1. Establish murine L929 fibroblast cell line cell monolayer by
plating 5 Â 10
6 cells in 20 mL of pre-warmed (37
C) RPMIFBS per 75 cm
2 flask or 10 Â 10
6 cells in 40 mL of pre-warmed
RPMI-FBS per 175 cm
2 flask (see Note 3).
2. Incubate 3–4 days at 37
C, 5% CO 2 .
3. Remove the medium and carefully wash the monolayer twice
using pre-warmed (37
C) DPBS.
4. Incubate cells with 3 mL (75 cm
2 flasks) or 6 mL (175 cm
2
flasks) of Trypsin-EDTA solution for 10 min at 37
C and 5%
CO 2 .
5. Detach cells from the flask substrate by tapping and use a
scraper, if needed.
6. Rinse the flask with 10 mL or 20 mL of pre-warmed RPMIFBS and collect cell suspension in a sterile 50-mL
centrifuge tube.
7. Pellet cells by centrifugation at 220 Â g for 5 min at room
temperature.
8. Resuspend cells in 10 mL of pre-warmed RPMI-FBS.
9. Count cells using a counting chamber (expect to obtain
20 Â 10
6 cells/mL per 75 cm
2 flask).
High-Content Analysis of Host-Pathogen Interactions
667
using pre-warmed (37
C) DPBS.
4. 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 .
5. Detach cells from the flask substrate by tapping and use a
scraper, if needed.
6. Rinse the flask with 10 mL or 20 mL of pre-warmed RPMIFBS and collect cell suspension in a sterile 50-mL
centrifuge tube.
7. Pellet cells by centrifugation at 220 Â g for 5 min at room
temperature.
8. Resuspend cells in 10 mL of pre-warmed RPMI-FBS.
9. Count cells using a counting chamber (expect to obtain
10–20 Â 10
6 cells/mL per 75 cm
2 flask).
10. Use cells for any phenotypic assay (see Subheadings 3.11–
3.24).
11. Repeat steps 1–9 to maintain and passage A549 cells twice a
week, for a maximum of ten passages, or pellet and dilute cells
in FBS supplemented with 10% DMSO to reach a 5 Â 10
6
cells/mL suspension for long-term storage in liquid nitrogen
(see Note 3).
3.5 Production
of L929-Derived M-CSF
Conditioned Medium
for Murine Primary
Macrophage
Differentiation
and Culture [17]
1. Establish murine L929 fibroblast cell line cell monolayer by
plating 5 Â 10
6 cells in 20 mL of pre-warmed (37
C) RPMIFBS per 75 cm
2 flask or 10 Â 10
6 cells in 40 mL of pre-warmed
RPMI-FBS per 175 cm
2 flask (see Note 3).
2. Incubate 3–4 days at 37
C, 5% CO 2 .
3. Remove the medium and carefully wash the monolayer twice
using pre-warmed (37
C) DPBS.
4. Incubate cells with 3 mL (75 cm
2 flasks) or 6 mL (175 cm
2
flasks) of Trypsin-EDTA solution for 10 min at 37
C and 5%
CO 2 .
5. Detach cells from the flask substrate by tapping and use a
scraper, if needed.
6. Rinse the flask with 10 mL or 20 mL of pre-warmed RPMIFBS and collect cell suspension in a sterile 50-mL
centrifuge tube.
7. Pellet cells by centrifugation at 220 Â g for 5 min at room
temperature.
8. Resuspend cells in 10 mL of pre-warmed RPMI-FBS.
9. Count cells using a counting chamber (expect to obtain
20 Â 10
6 cells/mL per 75 cm
2 flask).
High-Content Analysis of Host-Pathogen Interactions
667