Infinite 200 pro system

Stabilized PDCs were seeded in 384-well plates (1000 cells/20 μl/well) in quadruplicate for each treatment. A total of 16 or 48 compounds were used for screening each PDC sample (Additional file 2: Table S1, S2). After overnight incubation, the cells were treated with one drug at a 5-fold serial dilution for a total of 6 doses (50 μM ~ 16 nM). Cell viability was measured after 72 hrs of treatment using the CellTiter-Glo Luminescent Cell Viability Assay kit (Promega, Madison, WI, USA) and an Infinite 200 Pro system (TECAN, Mannedorf, Switzerland). Each screening plate contained a dimethyl sulfoxide (DMSO)-only vehicle to calculate relative cell viability and normalize the data. Dose response curve (DRC) fitting and area under the curve (AUC) values were assessed using GraphPad Prism 5.3 (GraphPad Software Inc., San Diego, CA, USA). A screening compound library was newly prepared every month and tested for the preservation of chemical activities using NSCLC cancer cell lines (A549, PC9 and H1299). All library compounds were purchased from Selleckchem (Houston, TX, USA).

For each treatment, stabilized PDCs were seeded in 384-well plates (1000 cells/well in 20 μL aliquots) in quadruplicate. Simultaneous tests were conducted for 64 chemotherapeutic agents that are commonly used to treat lung cancer or have shown promising efficacy in preclinical studies (Table S1). After overnight incubation, the cells were treated with one drug at a 5-fold serial dilution for a total of six doses (50 μM to 16 nM). Cell viability was measured after 72 h of treatment using the CellTiter-Glo Luminescent cell viability assay kit (Promega, Madison, WI, USA) and an Infinite 200 Pro system (TECAN, Mannedorf, Switzerland). Each screening plate contained a dimethyl-sulfoxide-only vehicle control to calculate the relative cell viability and normalize the data. Dose–response curve fitting and area under the curve (AUC) values were assessed using GraphPad Prism 5.3 (GraphPad Software Inc., San Diego, CA, USA). The relative sensitivity of each drug was determined by calculating standardized AUCs based on means and standard deviations, using Excel (Microsoft Office 2013 Excel).

Liposomes were synthesized as described previously (Mathur et al, 2019 (link)) and left untreated or treated with 1.5 Units/ml of lecithinase, the solvent containing lecithinase (ddH₂O) or heat inactivated lecithinase (treated at 100°C for 10 min), or BSA (1 μg/ml), or treated with Alexa Fluor 568 labeled lecithinase for 1 h at 4°C. The liposomes were sonicated at 100 amplitude for 5 min as control (CTRL). The released dye was captured by a cation exchanger resin Dowex (10–15 mg per well). The absorbance (OD) of residual dye was measured at a wavelength of 595 nm using the Infinite 200 PRO system (Tecan).

The analytical apparatus included a JEM-1200EX transmission electron microscopy (NEC Co), a Scanning electron microscope (SEM, Hitachi S4800), a Confocal laser scanning microscopy (Zeiss Co.), an XPert PRO X-ray Diffractometer (PANalytical Co.), a PPMS-9 vibration sample magnetometer (Quantum Design Co.), a Jasco-4100 Fourier transform infrared spectroscopy (Jasco Co.), a Waters SYNAPT G2 quadrupole-ion mobility-time-of-flight mass spectrometry (Waters MS Technologies Co.), a PB-10 Sartorius pH meter (Sartorius Co.), a Tecan's Infinite 200 PRO system (Tecan Co.), and a Milli-Q Water Purification System (Millipore Co.).

Bacterial strains were grown in ABT minimal medium^{64 (link)} supplemented with 5 g/L glucose and 2 g/L casamino acids (ABTGC), with or without addition of 8 μg/mL AZM (Spectrum). The supernatants of overnight culture were filter sterilized before quantification of elastase and rhamnolipid production. Elastase activity was measured using EnzCheck Elastase Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. The quantification of rhamnolipid was performed as previously described^{65 (link)} with modifications. Briefly, rhamnolipid was extracted from 1 mL of filtered supernatants with 2 mL of diethyl ether. Then, 1 mL of the diethyl ether extract was collected, vacuum dried and re-dissolved in 50 μL of deionized water. The solution was added with 450 μL of 0.19% (w/v) orcinol dissolved in 53% H₂SO₄, followed by heating at 80 °C for 30 min. The elastase activity and rhamnolipid production were quantified by measuring emission at 530 nm upon excitation at 485 nm and absorbance at 421 nm by using an Infinite 200 PRO system (Tecan), respectively. The results were normalized with optical density at 600 nm (OD₆₀₀) of the bacterial culture. Both elastase and rhamnolipid quantification assays were successfully replicated for at least three times, with measurements from distinct samples shown in the figure.

For cell viability assay, MCF-7 and ZR75-1 cells treated with siRNA scramble and siLINC00504 were plated in 96 well plates. A total of 500 cells were seeded in each well. The cell viability was evaluated after 48, 72, and 96 h of transfection. On each day, the media were replaced with 100 µL of fresh media + 10 µL Resazurin dye (7-hydroxy-3H-phenoxazin-3-one 10-oxide) at concentration of 0.1 mg/mL and incubated for 4 h, and read at 600 ηm and 570 ηm using TECAN Infinite^® 200 PRO system. The experiment was made in triplicate.