2.4 Measuring
Growth by ATP
1. 96- or 384-well, flat bottom white plates.
2. BacTiter-Glo™ Microbial Cell viability Assay (Promega).
3. Luminometer, e.g., Spectramax M5.
2.5 Measuring
Growth by Optical
Density
1. 96- or 384- well, flat clear bottom plates.
2. Spectrometer, e.g., Spectramax M5.
2.6 Measuring
Intracellular MICs
1. M. tuberculosis H37Rv:luc [3], M. tuberculosis H37Rv containing the Photinus pyralis luciferase gene (Hygromycin resistant
plasmid).
2. Monocytes (THP1) (ATCC
® TIB-202™).
3. 7H9-ADC-Tyloxapol (see Subheading 2.2, item 2).
4. 4 mm glass beads.
5. 4Â 50 mL conical tubes and centrifuge with rotor for these
tubes.
6. RPMI Complete medium: RPMI 1640 HEPES modification,
2 mM L-glutamine, 1 mM sodium pyruvate, 10% (v/v) fetal
bovine serum.
7. RPMI plus 0.05%(v/v) Tyloxapol.
8. 490 cm
2 roller bottle.
9. RPMI plus PMA: add 20 nM of Phorbol 12-myristate 13-acetate (PMA).
10. Sterile 384-well white plate with tissue culture treated surface.
11. Bright-Glo™ Luciferase Assay System (Promega).
12. Luminometer.
2.7 Calculation of %
Inhibition, MIC, pIC 50 ,
and IC 50
1. Analysis software for 96/384-well plates, e.g., Grafit 5 (Erithacus Software Limited), GraphPad Prism, or ActivityBase
(ID Business Solutions Limited).
3 Methods
3.1 Plate Preparation
1. To test the compounds starting at the highest concentration of
125 μM, dispense 50 μL of stock solution of each compound
(see Note 5) in Column 1 and 13 of V-bottom 384-well plates
(or in column 1 of 96-well plates) (see Note 6). Note the
dilution factor will be 1:100 for the final assay plates (see
Notes 7 and 8). Use the particular plate type specified in
Subheading 2 for the readout you will be using.
2. Add 25 μL DMSO to columns 2–5, 7–12, 14–17 and 19–24 in
384-well plates (or to columns 2–10 in 96-well plates). Do not
Antimicrobial Susceptibility Testing
641
Growth by ATP
1. 96- or 384-well, flat bottom white plates.
2. BacTiter-Glo™ Microbial Cell viability Assay (Promega).
3. Luminometer, e.g., Spectramax M5.
2.5 Measuring
Growth by Optical
Density
1. 96- or 384- well, flat clear bottom plates.
2. Spectrometer, e.g., Spectramax M5.
2.6 Measuring
Intracellular MICs
1. M. tuberculosis H37Rv:luc [3], M. tuberculosis H37Rv containing the Photinus pyralis luciferase gene (Hygromycin resistant
plasmid).
2. Monocytes (THP1) (ATCC
® TIB-202™).
3. 7H9-ADC-Tyloxapol (see Subheading 2.2, item 2).
4. 4 mm glass beads.
5. 4Â 50 mL conical tubes and centrifuge with rotor for these
tubes.
6. RPMI Complete medium: RPMI 1640 HEPES modification,
2 mM L-glutamine, 1 mM sodium pyruvate, 10% (v/v) fetal
bovine serum.
7. RPMI plus 0.05%(v/v) Tyloxapol.
8. 490 cm
2 roller bottle.
9. RPMI plus PMA: add 20 nM of Phorbol 12-myristate 13-acetate (PMA).
10. Sterile 384-well white plate with tissue culture treated surface.
11. Bright-Glo™ Luciferase Assay System (Promega).
12. Luminometer.
2.7 Calculation of %
Inhibition, MIC, pIC 50 ,
and IC 50
1. Analysis software for 96/384-well plates, e.g., Grafit 5 (Erithacus Software Limited), GraphPad Prism, or ActivityBase
(ID Business Solutions Limited).
3 Methods
3.1 Plate Preparation
1. To test the compounds starting at the highest concentration of
125 μM, dispense 50 μL of stock solution of each compound
(see Note 5) in Column 1 and 13 of V-bottom 384-well plates
(or in column 1 of 96-well plates) (see Note 6). Note the
dilution factor will be 1:100 for the final assay plates (see
Notes 7 and 8). Use the particular plate type specified in
Subheading 2 for the readout you will be using.
2. Add 25 μL DMSO to columns 2–5, 7–12, 14–17 and 19–24 in
384-well plates (or to columns 2–10 in 96-well plates). Do not
Antimicrobial Susceptibility Testing
641