Safire2 plate reader

For GFP reporter assays, 2-ml volumes of overnight Y. pestis cultures in TMH medium with 0.2% glucose were subcultured at an OD₆₂₀ of 0.1 into 2 ml of TMH medium incubated at 37°C in a rotary drum for 6 h. Cultures were diluted to an OD₆₂₀ of 0.25 to 0.40, and 200-μl aliquots were added in duplicate to a 96-well plate. Fluorescence was read on a Tecan Safire II plate reader, and results are reported as previously described (6 (link)).

Alamar Blue (Invitrogen, Waltham, MA, USA) was used to evaluate the overall metabolic status of ECB-NGF devices or NGC0211 cells. Briefly, NGC-0211 cells (1 × 10⁴ cells/well/100 µL) were seeded in clear bottom 96-well black plates (Corning, New York, NY, USA) for 24 h and exposed to control media or MCMs for 3 and 24 h, respectively. In the meantime, 10 µL of 10× Alamar Blue was added to every well during the last 1 h of treatment incubation. Fluorescence was then read in a spectrophotometer (Safire II Plate reader, Tecan, Männedorf, Switzerland; 5 nm bandpass; top read) at 560/590 nm. Similarly, post 7 weeks of experimentation with ECB-NGF devices, 50 µL of 10× Alamar Blue was added in 500 µL DMEM/F12 medium containing 5% FBS and incubated for 1 h. From each well, 100 µL was drawn in triplicate, plated in black bottom 96-well plate (Corning, New York, NY, USA) and the fluorescence was read as mentioned above.

A panel of 39 compounds against various cellular targets was obtained from Selleck Chemicals (Table S1 in File S1). Cells were seeded in 50 µl medium in 96-well plates at 8000 cells/well and incubated overnight. Serial dilutions of compounds were performed starting from 200 µM with 1: 4 dilutions for subsequent dilutions. Serially diluted compounds (50 µl) were added to cells and incubated for 48 hours. To measure cell viability, CellTiter-Glo (Promega) was added to the wells at 1∶1 ratio. After 10 minutes of incubation at room temperature, luminescence was measured with Safire II plate reader (Tecan). The experiments were carried out in triplicates for each dose dilution point and independently replicated on a separate occasion. Data was analyzed with Graphpad Prism software to determine the half maximal inhibitory concentration (IC₅₀). P-values were computed from Mann Whitney U-test.

Intracellular reactive oxygen species (ROS) generation and glutathione (GSH) extinction are early markers of stress, and play a major role in initiating the oxidative damage in cells. We used 20 µM dichlorodihydrofluorescein diacetate (DCFH-DA; excitation/emission—485/520 nm) to measure ROS and 50 µM monochlorobimane (mBCL; 394/490 nm) to measure GSH (Invitrogen, Waltham, MA, USA), respectively. For early time point measurements, NGC0211 cells (1 × 10⁴ cells/well/100 µL) were cultured for 24 h in clear bottom black 96-well plates (Corning, New York, NY, USA) and pre-incubated with either DCFH-DA or mBCL for 20 min. Cells were then treated with control media or MCMs and fluorescence kinetic readings were immediately started in a spectrophotometer (Safire II Plate reader, Tecan; 5 nm bandpass; bottom read) for the first one hour. The plates were then returned to an incubator maintained at 5% CO₂ and 37 °C and read after the completion of 3 h incubation. To study the effect after 24 h exposure, separate plates containing NGC0211 cells (1 × 10⁴ cells/well/100 µL) were adequately treated, DCFH-DA or mBCL was added 30 min prior to completion of treatment incubation time, and endpoint readings were obtained using the spectrophotometer, as mentioned above.

A total of 5 × 10⁴ neutrophils were seeded on a white 96-well flat-bottomed plate in calcium- and magnesium-positive media and rested at 37° for 1 h. Luminol (Sigma) (50 μM) and horseradish peroxidase (Sigma) (1.2 U/ml) were added followed by stimulation with PDBu (Sigma) (50 nM) or media. Luminescence was immediately read on a Safire II plate reader (Tecan).