(c) Plate dilutions on 7H11 agar plates and grow for
2–3 weeks at 37
C.
(d) Count CFU on plates from the different dilutions.
(e) Plot CFU as a function of RFU and fit the curve using a
linear regression to obtain parameters for the CFU ¼ f
(RFU) relationship. Same parameters can be reused for all
aliquots of the same stock, but a new regression should be
established for each stock of bacteria.
3.2 RAW264.7
Macrophage Cell Line
Culture
1. Establish RAW264.7 macrophage monolayer by plating
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 for 3–4 days at 37
C and 5% CO 2 .
3. Remove the medium and carefully wash the monolayer twice
using pre-warmed 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
20–40 Â 10
6 cells per 75 cm
2 flask).
10. Use cells for any phenotypic assay (see Subheadings 3.11–
3.24).
11. Repeat steps 1–9 twice a week to maintain and passage
RAW264.7 cells, 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).
3.3 THP-1
Macrophage Cell Line
Culture
1. Just after thawing, establish a THP-1 cell suspension at
0.5 Â 10
6 cells/mL in 20 mL of pre-warmed (37
C) RPMIFBS per 75 cm
2 flask (see Note 3).
2. Incubate for 4 days at 37
C, 5% CO 2 (incubate the flask
vertically).
3. Collect the cell suspension in a sterile 50-mL centrifuge tube.
High-Content Analysis of Host-Pathogen Interactions
665
2–3 weeks at 37
C.
(d) Count CFU on plates from the different dilutions.
(e) Plot CFU as a function of RFU and fit the curve using a
linear regression to obtain parameters for the CFU ¼ f
(RFU) relationship. Same parameters can be reused for all
aliquots of the same stock, but a new regression should be
established for each stock of bacteria.
3.2 RAW264.7
Macrophage Cell Line
Culture
1. Establish RAW264.7 macrophage monolayer by plating
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 for 3–4 days at 37
C and 5% CO 2 .
3. Remove the medium and carefully wash the monolayer twice
using pre-warmed 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
20–40 Â 10
6 cells per 75 cm
2 flask).
10. Use cells for any phenotypic assay (see Subheadings 3.11–
3.24).
11. Repeat steps 1–9 twice a week to maintain and passage
RAW264.7 cells, 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).
3.3 THP-1
Macrophage Cell Line
Culture
1. Just after thawing, establish a THP-1 cell suspension at
0.5 Â 10
6 cells/mL in 20 mL of pre-warmed (37
C) RPMIFBS per 75 cm
2 flask (see Note 3).
2. Incubate for 4 days at 37
C, 5% CO 2 (incubate the flask
vertically).
3. Collect the cell suspension in a sterile 50-mL centrifuge tube.
High-Content Analysis of Host-Pathogen Interactions
665