square petri dish or 2 Â 10
6 cells in 10 mL of RPMI-FBS
supplemented with GM-CSF per 78.5-cm
2 petri dish (see
Note 5).
2. Incubate cells for 4 days at 37
C, 5% CO 2 .
3. On day 4, add 50 mL per 500-cm
2 or 5 mL per 78.5-cm
2 petri
dish of fresh pre-warmed RPMI-FBS supplemented with
GM-CSF.
4. Incubate cells for 3 days more at 37
C, 5% CO 2 .
5. On day 7, repeat the steps 3 and 4.
6. On day 10, remove and discard the culture medium from the
petri dishes (using a 25 mL pipette).
7. Flush the cell monolayer by 5 aspiration/discharge cycles with
50 mL or 5 mL of pre-warmed (37
C) DPBS (using a 25 mL
pipette).
8. Remove and discard the supernatant.
9. Add 50 mL or 5 mL of pre-warmed (37
C) 2 mM EDTA
solution in DPBS.
10. Incubate cells for 5 min at 37
C, 5% CO 2 .
11. Flush the cell monolayer by 5 aspiration/discharge cycles with
50 mL or 5 mL of 2 mM EDTA in DPBS.
12. Collect cell suspension in a sterile 50-mL centrifuge tube.
13. Centrifuge at 220 Â g for 5 min and discard the supernatant.
14. Resuspend cells in 10 mL of pre-warmed RPMI-FBS.
15. Count cells using a counting chamber (expect to obtain
7 Â 10
7 cells per 500-cm
2 petri dish, corresponding to cell
number needed to prepare approximately four 384-well
plates).
16. Use for any phenotypic assay (see Subheadings 3.11–3.24).
3.10 Human Primary
Macrophage Cell
Culture
Human macrophages are CD14
+ differentiated peripheral blood
mononucleated cells (PBMC), isolated from healthy volunteer
peripheral blood samples.
1. Dilute blood (approximately 500 mL) or buffy coat (approximately 50 mL) 1:2 in pre-warmed (37
C) DPBS-FBS. Use a
sterile single-use beaker or sterile 50-mL tubes.
2. Distribute first 15 mL of Ficoll solution in 50-mL centrifuge
tubes and carefully deposit 30 mL of diluted blood on top of
Ficoll solution. Do not mix the tube.
3. Centrifuge at 850 Â g for 20 min using a rotor with swing-out
buckets, with braking speed of the centrifuge set to zero
(expect to obtain total duration centrifugation of 40 min).
High-Content Analysis of Host-Pathogen Interactions
671
6 cells in 10 mL of RPMI-FBS
supplemented with GM-CSF per 78.5-cm
2 petri dish (see
Note 5).
2. Incubate cells for 4 days at 37
C, 5% CO 2 .
3. On day 4, add 50 mL per 500-cm
2 or 5 mL per 78.5-cm
2 petri
dish of fresh pre-warmed RPMI-FBS supplemented with
GM-CSF.
4. Incubate cells for 3 days more at 37
C, 5% CO 2 .
5. On day 7, repeat the steps 3 and 4.
6. On day 10, remove and discard the culture medium from the
petri dishes (using a 25 mL pipette).
7. Flush the cell monolayer by 5 aspiration/discharge cycles with
50 mL or 5 mL of pre-warmed (37
C) DPBS (using a 25 mL
pipette).
8. Remove and discard the supernatant.
9. Add 50 mL or 5 mL of pre-warmed (37
C) 2 mM EDTA
solution in DPBS.
10. Incubate cells for 5 min at 37
C, 5% CO 2 .
11. Flush the cell monolayer by 5 aspiration/discharge cycles with
50 mL or 5 mL of 2 mM EDTA in DPBS.
12. Collect cell suspension in a sterile 50-mL centrifuge tube.
13. Centrifuge at 220 Â g for 5 min and discard the supernatant.
14. Resuspend cells in 10 mL of pre-warmed RPMI-FBS.
15. Count cells using a counting chamber (expect to obtain
7 Â 10
7 cells per 500-cm
2 petri dish, corresponding to cell
number needed to prepare approximately four 384-well
plates).
16. Use for any phenotypic assay (see Subheadings 3.11–3.24).
3.10 Human Primary
Macrophage Cell
Culture
Human macrophages are CD14
+ differentiated peripheral blood
mononucleated cells (PBMC), isolated from healthy volunteer
peripheral blood samples.
1. Dilute blood (approximately 500 mL) or buffy coat (approximately 50 mL) 1:2 in pre-warmed (37
C) DPBS-FBS. Use a
sterile single-use beaker or sterile 50-mL tubes.
2. Distribute first 15 mL of Ficoll solution in 50-mL centrifuge
tubes and carefully deposit 30 mL of diluted blood on top of
Ficoll solution. Do not mix the tube.
3. Centrifuge at 850 Â g for 20 min using a rotor with swing-out
buckets, with braking speed of the centrifuge set to zero
(expect to obtain total duration centrifugation of 40 min).
High-Content Analysis of Host-Pathogen Interactions
671