Secondary assay to identify inhibitors of non-replicating M. tb using luciferase expression without log phase outgrowth Measured in Microorganism System Using Plate Reader - 2157-04_Inhibitor_Dose_DryPowder_Activity_Set2
This assay determines the inhibitory effect of small molecules against M. tuberculosis under carbon starvation conditions ..more
BioActive Compounds: 52
Mycobacterium tuberculosis, drug resistance, non-replicating, carbon starvation
This assay determines the inhibitory effect of small molecules against M. tuberculosis under carbon starvation conditions
Compounds are designated as active if 90% inhibition of luminescence was observed relative to DMSO control wells.
1. 7H9 Complete (4.7 gm Middlebrook 7H9 powder, 900 mL water, 100 mL OADC (BD/Difco), 4 mL 50% glycerol, 2.5 mL 20% Tween-80)
2. 7H9/Tyloxapol: (4.7 gm Middlebrook 7H9 powder, 1000 mL water, 2.5 mL 20% tyloxapol)
Strain: M. tuberculosis H37Rv with an inducible firefly luciferase expression plasmid
5 weeks prior to the assay, a M. tuberculosis culture in logarithmic growth phase (OD600 between 0.4-0.8) was washed 2 times in 7H9/Tyloxapol media
and then resuspended in 7H9/Tyloxapol to an OD600 = 0.2. 50 mL of cells were then placed into a sterile roller bottle and incubated standing at 37 C for 5 weeks.
On the day of the assay, outside of the BSL3 a master plate containing a 2-fold dilution series of a small molecule in DMSO was created in 96 well format (top concentration 50 mM, bottom concentration DMSO only). 4uL of the master plate dilution series was transferred to the first row of an opaque 96 well assay plate. 196 uL of media was added to this row. After pipetting up and down to mix, 50 uL was transferred to the next three rows, so that the top four rows of the plate contain 50 uL of diluted small molecule in media. This process was repeated with a new small molecule in the bottom four rows of the assay plate. The assay plate was then brought into the BSL3.
Inside the BSL3, the starvation culture was diluted to an OD600 = 0.1 using 7H9/Tyloxapol media and anhydrotetracycline was added to a final concentration of 50 ng/mL. 50uL of the diluted TB culture was added to each well of the 96 well plate. The plate was incubated at 37 C in a sealed Tupperware container for 5 days containing a wet kimtech shop towel. After 5 days Cell Culture Lysis reagent (Promega #E1531) was added to the wells and mixed by pipetting. After a 10 minute incubation at room temperature, 100 uL of luciferase reagent (Promega, #E1500) was then added to the wells and luminescence immediately read using the M5 Spectramax (Molecular Devices) plate reader. The active concentration was defined as the concentration that results in 90% inhibition of luminescence relative to the DMSO controls.
All inactive compounds received an activity score of 1. The active compounds were ranked on a scale of 10-100, with 100 representing the most active compound tested in this assay.
Categorized Comment - additional comments and annotations
* Activity Concentration.
Data Table (Concise)