M200pro plate reader

Quantification of IFNγ and IL-6 were conducted using commercial (elisakit.com, 2018, Scoresby, Australia) enzyme-linked immunosorbent assays (ELISA) according to the manufacturer’s instructions. Cell lysates were diluted 1:1.5 (v/v) with assay diluent 1B (supplied) and samples (100 μL) or standards (100 μL, 0–1000 pg/mL IFNγ/IL-6) were loaded into pre-coated 96-well plates (supplied), sealed, and incubated for 2 h in a humidified chamber. Plates were then washed 4× and incubated with detection antibody (100 μL, 75 ng/mL (anti-IL-6) or 25 ng/mL (anti-IFNγ), prepared in assay diluent 1B) for 1 h. Plates were then washed 4× and incubated with streptavidin-horse radish peroxidase (HRP) conjugate (100 μL, diluted 1:500 v/v in assay diluent 1B) for 45 min, then washed 5× and further incubated in 100 μL of tetramethylbenzidine (TMB) substrate for 15 min (IL-6) or 20 min (IFNγ), protected from light. Reaction was stopped by addition of supplied solution (50 μL) and absorbance immediately determined at 450 nm (with wavelength correction of 570 nm) using a TECAN M200 PRO plate reader (Tecan, Melbourne, Australia). All data were normalised relative to the corresponding total protein.

M. tuberculosis or M. smegmatis was inoculated into 7H9 medium with or without the test compounds at an ODλ₆₀₀ of 0.1 and incubated with shaking at 37°C for 24 h (M. tuberculosis) or 12 h (M. smegmatis). An aliquot of the culture was heat inactivated at 95°C for 20 min and diluted 1:100. Diluted samples were mixed with the BacTiter-Glo assay (Promega) reagent at a 1:1 ratio, and luminescence was quantified on a Tecan M200 Pro plate reader (integration = 1 s). Relative luminescence units (RLU) were normalized to the number of log₁₀ CFU in the sample to account for differences in bacterial number.

Turbidity-based growth inhibition was performed to assess anti-mycobacterial potency of the compounds. M. bovis BCG or M. tuberculosis H37Rv pre-cultures were harvested at mid-log phase and diluted to OD₆₀₀ 0.05 in complete 7H9 media. Bacterial suspensions were then dispensed in 96-well plate (200 μL/well) in presence of two-fold serially diluted compounds and incubated for 5 days at 37°C under shaking (100 rpm). Cells were manually resuspended and OD was measured at 600 nm on M200Pro plate reader (Tecan). Percentage of growth was determined as compared to untreated control and plotted as a function of drug concentration. MIC₅₀ (i.e., the concentration that inhibit 50% of growth) was determined in three independent replicates. MBC was determined by colony forming unit (CFU) enumeration on agar. Briefly, 10⁶ CFU from mid-log phase pre-culture of M. tuberuculosis H37Rv were treated for 5 days with concentrations ranging from one to ten times the MIC₉₀ (Minimum inhibitory concentration required to inhibit 90% of bacterial growth) of each compound. Treated cultures were then plated on agar and CFU counted after 3–4 weeks of incubation at 37°C. CFU from an untreated inoculum were enumerated in similar manner as a control. MBC₉₉ (i.e., the concentration that induce a 100-fold reduction in CFU count) was recorded. MBC₉₉ was determined thrice independently.

Cell-based CT-like peptidase assay was performed using the Proteasome-Glo™ Cell-Based Assay Reagent (Promega) according to manufacturer's guidelines. Briefly, HepG2 cells (10⁴ cells/well) or M. bovis BCG log phase cultures were treated with the indicated compounds for 2 h followed by incubation with the luminescent substrate for 10 min. Luminescence was detected with a Tecan M200 Pro plate reader. Relative luminescence (RLU) was plotted as a function of drug concentrations and Proteasome IC₅₀ (i.e., the concentration required to inhibit 50% of the proteasome activity) was determined. Bortezomib was used as a positive control.

The assay was used to quantify the effect of PF74 derivatives on the assembly of mature-like HIV-1 particles, as described in [58 (link)]. In 96-well plate, we pre-incubated the HIV-1 CANC protein (60 µg/100 µL) with the tested inhibitor (final concentration 10 µM) and kept it on ice for 1 h. To start the assembly reaction, 3 µg of dually labelled oligonucleotide (tqON) was added to the CANC protein and the volume of the reaction mixture was adjusted to 100 µL using the assembly buffer (50 mM Tris, pH 8.0, 1 µM ZnCl₂, 340 mM NaCl). Following a 3-h incubation at room temperature, Exonuclease I (ExoI) 6 mM Mg²⁺ was added and the fluorescence of the fluorophore released from degraded tqON was measured using a Tecan M200Pro plate reader.