11. Repeat twice steps 8–10 to make sure that the supernatant
appears clear after three washings.
12. Centrifuge at 3750 Â g for 5 min at room temperature.
13. Resuspend the bacterial pellet in 1 mL of RPMI-FBS.
14. Wait 5 min at room temperature to pellet clumped bacteria and
use homogeneous supernatant for infection (see Note 17).
15. Calculate the bacterial concentration by measuring the optical
density (O.D.) and the fluorescence (RFU) of the bacterial
stock suspension using a spectrophotometer and the microplate fluorescence reader, respectively. Determine the bacterial
concentration referring to the pre-established relationship,
such as 1-unit O.D. is corresponding to 10
8 CFU/mL bacteria
concentration, and the CFU ¼ f(RFU) linear relationship previously established for the used fluorescent bacterial stock
(as described in Subheading 3.1).
16. Remove the supernatant from the plates using a multichannel
pipette.
17. In dedicated wells for cell infection: add 50 μL per well of
pHrodo-labeled bacteria at a MOI of 2 in RPMI-FBS (see
Note 18).
18. In dedicated empty wells (without cells) for positive controls
(intracellular pH indicator controls): add first 50 μL per well of
CO 2 -independent Medium to a pH range from 4 to 10, and
then 5 μL of the previously diluted pHrodo-labeled bacteria in
RPMI-FBS to reach a MOI of 2 (see Note 18), to determine a
pHrodo standard curve relative intensity per bacteria.
19. Seal plates using breathable sealing films and centrifuge
220 Â g for 1 min at room temperature.
20. Incubate plates for 24 h at 37
C with 5% CO 2 .
21. Remove the breathable sealing films.
22. Remove the supernatant from the plates using a multichannel
pipette.
23. Wash cells three times with 50 μL per well of DPBS using a
multichannel pipette.
24. Add 50 μL per well of RPMI-FBS containing 10 μg/mL
Hoechst.
25. Incubate plates for 30 min at 37
C with 5% CO 2 .
26. Repeat steps 22 and 23 three times with RPMI-FBS.
27. Immediately proceed to image the cells in 50 μL per well of
RPMI-FBS, using the high-content confocal microscope (see
Subheading 3.25). Use 60Â magnification. Plates cannot be
stored.
High-Content Analysis of Host-Pathogen Interactions
685
appears clear after three washings.
12. Centrifuge at 3750 Â g for 5 min at room temperature.
13. Resuspend the bacterial pellet in 1 mL of RPMI-FBS.
14. Wait 5 min at room temperature to pellet clumped bacteria and
use homogeneous supernatant for infection (see Note 17).
15. Calculate the bacterial concentration by measuring the optical
density (O.D.) and the fluorescence (RFU) of the bacterial
stock suspension using a spectrophotometer and the microplate fluorescence reader, respectively. Determine the bacterial
concentration referring to the pre-established relationship,
such as 1-unit O.D. is corresponding to 10
8 CFU/mL bacteria
concentration, and the CFU ¼ f(RFU) linear relationship previously established for the used fluorescent bacterial stock
(as described in Subheading 3.1).
16. Remove the supernatant from the plates using a multichannel
pipette.
17. In dedicated wells for cell infection: add 50 μL per well of
pHrodo-labeled bacteria at a MOI of 2 in RPMI-FBS (see
Note 18).
18. In dedicated empty wells (without cells) for positive controls
(intracellular pH indicator controls): add first 50 μL per well of
CO 2 -independent Medium to a pH range from 4 to 10, and
then 5 μL of the previously diluted pHrodo-labeled bacteria in
RPMI-FBS to reach a MOI of 2 (see Note 18), to determine a
pHrodo standard curve relative intensity per bacteria.
19. Seal plates using breathable sealing films and centrifuge
220 Â g for 1 min at room temperature.
20. Incubate plates for 24 h at 37
C with 5% CO 2 .
21. Remove the breathable sealing films.
22. Remove the supernatant from the plates using a multichannel
pipette.
23. Wash cells three times with 50 μL per well of DPBS using a
multichannel pipette.
24. Add 50 μL per well of RPMI-FBS containing 10 μg/mL
Hoechst.
25. Incubate plates for 30 min at 37
C with 5% CO 2 .
26. Repeat steps 22 and 23 three times with RPMI-FBS.
27. Immediately proceed to image the cells in 50 μL per well of
RPMI-FBS, using the high-content confocal microscope (see
Subheading 3.25). Use 60Â magnification. Plates cannot be
stored.
High-Content Analysis of Host-Pathogen Interactions
685