3. Add ten glass beads to each tube and disperse the bacterial
pellet by shaking for 60 s.
4. Add 6 mL of fresh RPMI media and let stand for 5 min at room
temperature (see Note 23).
5. Carefully collect 5 mL of the supernatant.
6. Pool all the supernatants into a single sterile tube and centrifuge at 750 Â g for 5 min to remove any remaining clumps;
save the supernatant as the dispersed bacterial culture and
discard the pellet.
7. Measure OD 600 of the bacterial culture using a 1:3 or 1:4
dilution in RPMI-0.05% Tyloxapol.
8. Calculate the volume needed to have a multiplicity of infection
(MOI) of 1. After the single cell preparation, we could use the
following conversion: An OD of 0.1 ¼ 1 Â 10
8 CFU/mL (see
Notes 12 and 25).
9. Culture THP1 cells in complete RPMI-1640 and incubated at
37
C with 5% CO 2 .
10. Infect 100 mL of THP1 Monocytes (4 Â 10
5 cell/mL) for 4 h
in a 490 cm
2 roller bottle at a MOI of 1 in RPMI-PMA.
11. Remove excess bacteria by washing five times in complete
RPMI (5 Â 750 Â g, 5 min).
12. Dispense 50 μL/well (10,000 cells/well) of infected THP1
cells in 384-well white plates with compound (see Subheading
3.1).
13. Incubate for 5 days at 37
C with 5% CO 2 .
14. Add 25 μL of reconstituted Bright-Glo™ to each well.
15. Incubate at room temperature for 30 min.
16. Measure luminescence (see Notes 24 and 25).
3.7 Calculation of %
Inhibition, MIC, pIC 50
and IC 50
The activity of a compound can be shown in different ways.
1. Calculate the % Inhibition ¼ 100 Â [(data À control1)/(control2 À control1)] and also % Remaining ¼ 100 À % Inhibition.
Control 1 is no inhibition and control 2 is complete inhibition
of bacterial growth.
2. Calculate the MIC as the lowest concentration of the compound that inhibits at least 90% of the growth (MIC 90 ) or 50%
of the growth (MIC 50 ).
3. Determine the pIC 50 or IC 50 as a nonlinear regression curve fit
with programs like Grafit 5, GraphPad Prism or ActivityBase.
644
Esther Pe ´ rez-Herra ´ n and Alfonso Mendoza
pellet by shaking for 60 s.
4. Add 6 mL of fresh RPMI media and let stand for 5 min at room
temperature (see Note 23).
5. Carefully collect 5 mL of the supernatant.
6. Pool all the supernatants into a single sterile tube and centrifuge at 750 Â g for 5 min to remove any remaining clumps;
save the supernatant as the dispersed bacterial culture and
discard the pellet.
7. Measure OD 600 of the bacterial culture using a 1:3 or 1:4
dilution in RPMI-0.05% Tyloxapol.
8. Calculate the volume needed to have a multiplicity of infection
(MOI) of 1. After the single cell preparation, we could use the
following conversion: An OD of 0.1 ¼ 1 Â 10
8 CFU/mL (see
Notes 12 and 25).
9. Culture THP1 cells in complete RPMI-1640 and incubated at
37
C with 5% CO 2 .
10. Infect 100 mL of THP1 Monocytes (4 Â 10
5 cell/mL) for 4 h
in a 490 cm
2 roller bottle at a MOI of 1 in RPMI-PMA.
11. Remove excess bacteria by washing five times in complete
RPMI (5 Â 750 Â g, 5 min).
12. Dispense 50 μL/well (10,000 cells/well) of infected THP1
cells in 384-well white plates with compound (see Subheading
3.1).
13. Incubate for 5 days at 37
C with 5% CO 2 .
14. Add 25 μL of reconstituted Bright-Glo™ to each well.
15. Incubate at room temperature for 30 min.
16. Measure luminescence (see Notes 24 and 25).
3.7 Calculation of %
Inhibition, MIC, pIC 50
and IC 50
The activity of a compound can be shown in different ways.
1. Calculate the % Inhibition ¼ 100 Â [(data À control1)/(control2 À control1)] and also % Remaining ¼ 100 À % Inhibition.
Control 1 is no inhibition and control 2 is complete inhibition
of bacterial growth.
2. Calculate the MIC as the lowest concentration of the compound that inhibits at least 90% of the growth (MIC 90 ) or 50%
of the growth (MIC 50 ).
3. Determine the pIC 50 or IC 50 as a nonlinear regression curve fit
with programs like Grafit 5, GraphPad Prism or ActivityBase.
644
Esther Pe ´ rez-Herra ´ n and Alfonso Mendoza