4. Pellet cells by centrifugation at 220 Â g for 5 min at room
temperature.
5. Resuspend cells in 10 mL of pre-warmed RPMI-FBS.
6. Count cells using a counting chamber (expect to obtain
20 Â 10
6 cells in 20 mL per 75 cm
2 flask) and adjust to
0.2–0.4 Â 10
6 cells/mL in maximum 40 mL per 75 cm
2 flask
and 80 mL per 175 cm
2 flask.
7. Repeat steps 2–6 to maintain and passage undifferentiated
THP-1 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).
8. To differentiate cells into macrophages for infection assays,
adjust to 20 Â 10
6 cells in 20 mL of pre-warmed RPMI-FBS
per 75 cm
2 flask or 40 Â 10
6 cells in 40 mL of pre-warmed
RPMI-FBS per 175 cm
2 flask.
9. Directly add 50 μg/mL PMA stock solution 1:1000 diluted in
the flask to obtain a final concentration of 50 ng/mL.
10. Incubate for 3 days at 37
C, 5% CO 2 (incubate the flask
horizontally).
11. Remove the medium and carefully wash the monolayer twice
using pre-warmed (37
C) DPBS.
12. 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 .
13. Detach cells from the flask substrate by tapping and use a
scraper, if needed.
14. Rinse the flask with 10 mL or 20 mL of pre-warmed RPMIFBS and collect cell suspension in a sterile centrifuge
50-mL tube.
15. Pellet cells by centrifugation at 220 Â g for 5 min at room
temperature.
16. Resuspend cells in 10 mL of pre-warmed RPMI-FBS.
17. Count cells using a counting chamber (expect to obtain
10 Â 10
6 cells per 75 cm
2 flask, corresponding to cell number
needed to prepare one 384-well plate).
18. Use for any phenotypic assay (see Subheadings 3.11–3.24).
3.4 A549 Human
Type II
Pneumocyte-Like Cell
Line Culture
1. Establish A549 human type II pneumocyte-like cell monolayer
by plating 0.5–1 Â 10
6 cells in 20 mL of pre-warmed (37
C)
RPMI-FBS per 75 cm
2 flask or 2 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 .
666
Nathalie Deboosere et al.
temperature.
5. Resuspend cells in 10 mL of pre-warmed RPMI-FBS.
6. Count cells using a counting chamber (expect to obtain
20 Â 10
6 cells in 20 mL per 75 cm
2 flask) and adjust to
0.2–0.4 Â 10
6 cells/mL in maximum 40 mL per 75 cm
2 flask
and 80 mL per 175 cm
2 flask.
7. Repeat steps 2–6 to maintain and passage undifferentiated
THP-1 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).
8. To differentiate cells into macrophages for infection assays,
adjust to 20 Â 10
6 cells in 20 mL of pre-warmed RPMI-FBS
per 75 cm
2 flask or 40 Â 10
6 cells in 40 mL of pre-warmed
RPMI-FBS per 175 cm
2 flask.
9. Directly add 50 μg/mL PMA stock solution 1:1000 diluted in
the flask to obtain a final concentration of 50 ng/mL.
10. Incubate for 3 days at 37
C, 5% CO 2 (incubate the flask
horizontally).
11. Remove the medium and carefully wash the monolayer twice
using pre-warmed (37
C) DPBS.
12. 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 .
13. Detach cells from the flask substrate by tapping and use a
scraper, if needed.
14. Rinse the flask with 10 mL or 20 mL of pre-warmed RPMIFBS and collect cell suspension in a sterile centrifuge
50-mL tube.
15. Pellet cells by centrifugation at 220 Â g for 5 min at room
temperature.
16. Resuspend cells in 10 mL of pre-warmed RPMI-FBS.
17. Count cells using a counting chamber (expect to obtain
10 Â 10
6 cells per 75 cm
2 flask, corresponding to cell number
needed to prepare one 384-well plate).
18. Use for any phenotypic assay (see Subheadings 3.11–3.24).
3.4 A549 Human
Type II
Pneumocyte-Like Cell
Line Culture
1. Establish A549 human type II pneumocyte-like cell monolayer
by plating 0.5–1 Â 10
6 cells in 20 mL of pre-warmed (37
C)
RPMI-FBS per 75 cm
2 flask or 2 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 .
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Nathalie Deboosere et al.