10. Repeat steps 1–9 to maintain and passage L929 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).
11. To produce M-CSF-enriched supernatant, adjust to
7 Â 10
6 L929 cells in 70 mL of pre-warmed RPMI-FBS per
175 cm
2 flask.
12. Incubate for 7–8 days at 37
C, 5% CO 2 .
13. Collect and pool the cell supernatants in sterile 50-mL centrifuge tubes.
14. Pellet cells by centrifugation at 220 Â g for 5 min at room
temperature.
15. Filter-sterilize the cell supernatants through a 0.22 μm polyether sulfone membrane.
16. Store 10 mL and 50 mL aliquots at À20
C and avoid
refreezing.
17. Determine the M-CSF concentration of the L929-culture
supernatants to be used to obtain differentiation of monocytes
into macrophages (see Subheading 3.8 and Note 6).
3.6 Production
of J558-Derived
GM-CSF Conditioned
Medium for Murine
Primary Dendritic Cell
Differentiation
and Culture [18]
1. Establish murine J558 cell line cell monolayer by plating
5 Â 10
6 cells in 10 mL of pre-warmed (37
C) IMDM-FBS
supplemented with Geneticin per 25 cm
2 flask (see Note 3).
2. Incubate for 2 days at 37
C, 5% CO 2 .
3. Collect the cell supernatant and transfer into a new 25 cm
2
flask.
4. Gently replace 10 mL of freshly pre-warmed IMDM-FBS supplemented with Geneticin in the first 25 cm
2 flask containing
adherent cells.
5. Incubate the two 25 cm
2 flasks for 2 days at 37
C, 5% CO 2 .
6. Detach adherent cells by using a scraper and resuspend in the
cell supernatants (expect to obtain total 2 Â 10
6 cells).
7. Collect and pool the cell suspensions obtained from the two
25 cm
2 flasks in sterile 50-mL centrifuge tubes.
8. Pellet cells by centrifugation at 220 Â g for 10 min at room
temperature.
9. Seed 2 Â 10
6 cells in 20 mL of pre-warmed IMDM-FBS
supplemented with Geneticin per 75 cm
2 flask.
10. Incubate 2–3 days at 37
C, 5% CO 2 .
11. Detach adherent cells from the flask substrate by using a
scraper and resuspend in the cell supernatants (expect to obtain
2 Â 10
7 cells per 75 cm
2 flask).
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Nathalie Deboosere et al.
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).
11. To produce M-CSF-enriched supernatant, adjust to
7 Â 10
6 L929 cells in 70 mL of pre-warmed RPMI-FBS per
175 cm
2 flask.
12. Incubate for 7–8 days at 37
C, 5% CO 2 .
13. Collect and pool the cell supernatants in sterile 50-mL centrifuge tubes.
14. Pellet cells by centrifugation at 220 Â g for 5 min at room
temperature.
15. Filter-sterilize the cell supernatants through a 0.22 μm polyether sulfone membrane.
16. Store 10 mL and 50 mL aliquots at À20
C and avoid
refreezing.
17. Determine the M-CSF concentration of the L929-culture
supernatants to be used to obtain differentiation of monocytes
into macrophages (see Subheading 3.8 and Note 6).
3.6 Production
of J558-Derived
GM-CSF Conditioned
Medium for Murine
Primary Dendritic Cell
Differentiation
and Culture [18]
1. Establish murine J558 cell line cell monolayer by plating
5 Â 10
6 cells in 10 mL of pre-warmed (37
C) IMDM-FBS
supplemented with Geneticin per 25 cm
2 flask (see Note 3).
2. Incubate for 2 days at 37
C, 5% CO 2 .
3. Collect the cell supernatant and transfer into a new 25 cm
2
flask.
4. Gently replace 10 mL of freshly pre-warmed IMDM-FBS supplemented with Geneticin in the first 25 cm
2 flask containing
adherent cells.
5. Incubate the two 25 cm
2 flasks for 2 days at 37
C, 5% CO 2 .
6. Detach adherent cells by using a scraper and resuspend in the
cell supernatants (expect to obtain total 2 Â 10
6 cells).
7. Collect and pool the cell suspensions obtained from the two
25 cm
2 flasks in sterile 50-mL centrifuge tubes.
8. Pellet cells by centrifugation at 220 Â g for 10 min at room
temperature.
9. Seed 2 Â 10
6 cells in 20 mL of pre-warmed IMDM-FBS
supplemented with Geneticin per 75 cm
2 flask.
10. Incubate 2–3 days at 37
C, 5% CO 2 .
11. Detach adherent cells from the flask substrate by using a
scraper and resuspend in the cell supernatants (expect to obtain
2 Â 10
7 cells per 75 cm
2 flask).
668
Nathalie Deboosere et al.