12. Collect and pool the cell suspensions obtained from the flasks
in sterile 50-mL centrifuge tubes.
13. Pellet cells by centrifugation at 220 Â g for 10 min at room
temperature.
14. Seed 7 Â 10
6 cells in 40 mL of pre-warmed IMDM-FBS
supplemented with Geneticin per 175 cm
2 flask.
15. Incubate 2 days more at 37
C, 5% CO 2 .
16. On day 8–9, repeat steps 11–13.
17. Resuspend cells in adapted volume of pre-warmed IMDMFBS.
18. Count cells using a counting chamber (expect to obtain
3 Â 10
7 cells/mL per 175 cm
2 flask).
19. Repeat steps 14–16 to maintain and passage J558 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).
20. To produce GM-CSF, adjust to 3.5 Â 10
6 J558 cells in 70 mL
of pre-warmed IMDM-FBS (without Geneticin) per 175 cm
2
flask.
21. Incubate for 6–7 days at 37
C, 5% CO 2 .
22. Collect and pool the cell supernatants.
23. Pellet cells by centrifugation at 220 Â g for 5 min at room
temperature.
24. Filter-sterilize the cell supernatants through a 0.22 μm polyethersulfone membrane.
25. Store 10 mL and 50 mL aliquots at À20
C and avoid
refreezing.
26. Determine the GM-CSF concentration of the J558-culture
supernatant to be used to obtain differentiation of hematopoietic stem cells into dendritic cells (Subheading 3.9 and
Note 7).
3.7 Murine Primary
Monocyte Isolation
Murine macrophages and dendritic cells are differentiated from
mice bone marrow.
1. Collect and pool bone marrow from at least one mouse in
50 mL of cold (4
C) RPMI-FBS in sterile 50-mL centrifuge
tubes.
2. Centrifuge at 220 Â g for 5 min and discard the supernatant.
3. Resuspend cells in 1 mL of 1Â Red blood cell lysis buffer and
incubate for exactly 1 min at room temperature.
4. Immediately dilute with 49 mL of cold RPMI-FBS.
High-Content Analysis of Host-Pathogen Interactions
669
in sterile 50-mL centrifuge tubes.
13. Pellet cells by centrifugation at 220 Â g for 10 min at room
temperature.
14. Seed 7 Â 10
6 cells in 40 mL of pre-warmed IMDM-FBS
supplemented with Geneticin per 175 cm
2 flask.
15. Incubate 2 days more at 37
C, 5% CO 2 .
16. On day 8–9, repeat steps 11–13.
17. Resuspend cells in adapted volume of pre-warmed IMDMFBS.
18. Count cells using a counting chamber (expect to obtain
3 Â 10
7 cells/mL per 175 cm
2 flask).
19. Repeat steps 14–16 to maintain and passage J558 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).
20. To produce GM-CSF, adjust to 3.5 Â 10
6 J558 cells in 70 mL
of pre-warmed IMDM-FBS (without Geneticin) per 175 cm
2
flask.
21. Incubate for 6–7 days at 37
C, 5% CO 2 .
22. Collect and pool the cell supernatants.
23. Pellet cells by centrifugation at 220 Â g for 5 min at room
temperature.
24. Filter-sterilize the cell supernatants through a 0.22 μm polyethersulfone membrane.
25. Store 10 mL and 50 mL aliquots at À20
C and avoid
refreezing.
26. Determine the GM-CSF concentration of the J558-culture
supernatant to be used to obtain differentiation of hematopoietic stem cells into dendritic cells (Subheading 3.9 and
Note 7).
3.7 Murine Primary
Monocyte Isolation
Murine macrophages and dendritic cells are differentiated from
mice bone marrow.
1. Collect and pool bone marrow from at least one mouse in
50 mL of cold (4
C) RPMI-FBS in sterile 50-mL centrifuge
tubes.
2. Centrifuge at 220 Â g for 5 min and discard the supernatant.
3. Resuspend cells in 1 mL of 1Â Red blood cell lysis buffer and
incubate for exactly 1 min at room temperature.
4. Immediately dilute with 49 mL of cold RPMI-FBS.
High-Content Analysis of Host-Pathogen Interactions
669