Infinite 500

For luminometric analyses using a plate reader, transgenic HepG2 cells were seeded in most cases at 2 × 10⁵ cells/cm² 2 days before use. Because secreted G-Luc and C-Luc are very stable, each G-Luc-containing sample was collected at sequential time points and centrifuged at 1,000 rpm for 3 min; the resultant supernatant was stored at 4°C until the endpoint of the assay. Ten microliters of stored supernatant was transferred into each of three wells of a white 96-microwell plate for each sample (Nunc, Thermo Fisher Scientific). After addition of 10 μl of the substrate described before, luminescence (RLU) was measured using Infinite500 (TECAN), a filter-based multimode microplate reader.

Hepa1.6 cells were seeded at 8,000 cells per well in a volume of 15 μl, in 384-well plates. Compounds and were added from a 4x concentrated stock, diluted in culture medium, to the cells, reaching a total assay volume of 20 μl. Assays were typically performed 24 hours after incubation with medium or compounds. ATP levels were measured using Cell Titer Glo (Promega, Madison, WI, USA). Mitochondrial membrane potential (Δψm) was measured using the fluorescent tetramethylrhodamine methyl ester (TMRM) probe (T-668, Invitrogen, Paisley, UK). Briefly, cells were incubated in culture medium and TMRM was added to the culture medium at a final concentration of 200 nM for 45 minutes at 37°C before washing with 50 μl of PBS. The Alamar Blue assay (Life Technologies, Paisley, UK) was used as a marker for redox potential, following manufacturer protocols. ATP level, redox potential and TMRM assays were measured on a Tecan Infinite 500 (Tecan, Männedorf, Switzerland), and performed in duplicate.

Each bacterial strain to be measured was grown overnight in mineral salts medium (Hall, 1998 (link)) supplemented with 0.2% glucose and the appropriate antibiotics. On the day of the measurement, the cultures were diluted 20-fold in fresh medium, and dispensed into clear-bottomed black-walled 96-well plates (Costar Ref. 3603, Corning, NY, USA). Growth and measurement of the cultures took place in a TECAN Infinite 500 fluorescent reader, shaken and incubated at 37°C. Optical density was determined at 600 nm. Fluorescence intensity was recorded using an excitation wavelength of 580 ± 10 nm, and an emission wavelength of 610 ± 5 nm, with a gain set to 40. Readings were taken every 12 min. Antibiotics were used at the following concentrations: ampicillin (Ap): 50 μg/ml, chloramphenicol (Cm): 25 μg/ml, kanamycin (Km): 30 μg/ml. Na-malonate was administered at 2 mg/ml, cerulenin was used at 20 μg/ml, and β-alanine at 3 mM end concentrations. For characterization of the sensors’ response to malonyl-CoA, IPTG was administered in a concentration-series of 0.01, 0.1, 0.3, 0.6, 1, and 10 mM. Arabinose was used in a log10 series from 10⁻⁴ to 10⁻¹% during optimization and at 10⁻²% for comparison of alternate malonyl-CoA producer constructs. All chemicals were obtained from SIGMA (St. Louis, MO, USA).

A cell counting kit-8 (CCK-8) assay (Sigma) was used to measure the cytotoxicity of three drugs on HEK293T cells. 10,000 cells per well were pre-seeded into 96 well plates in 100 µl complete culture medium. The culture medium was changed into drug containing medium with drug concentrations of 0 µM (0.1% DMSO),1 µM, 5 µM and 10 µM. After 24 h drug incubation, 10 µl WST-8 solution was added to each well for 4 h incubation under standard conditions. The absorbance at 450 nm was determined by a multiplate reader (Tecan Infinite 500).

Gene expression was evaluated by TaqMan qPCR in the knee joint and the popliteal lymph node that drains the knee joint. Following euthanization, knees and popliteal lymph nodes were excised. Combined joint tissue from the synovial wall and articular surface (not exceeding 30 mg total) and the popliteal lymph nodes were homogenized with bead pulverization in Qiazol. RNA was extracted and purified using the RNeasy Plus mini kit from Qiagen and quantified using NanoQuant plate from Tecan in a microplate reader (Tecan Infinite 500, Tecan). The RNA was converted to cDNA using the iScript Synthesis kit from Bio-Rad (Hercules). Gene expression was calculated by the ΔΔCt method, normalizing to GAPDH and beta-actin (ACTB). TaqMan reagents were purchased from ThermoFisher and used according to provided protocols, using appropriate primers (IL-12β: Mm01288989_m1, IL-6: Mm01210732_g1, MMP13: Mm00439491_m1, TNF-α: Mm00443258_m1, GAPDH: Mm99999915_g1, ACTB: Mm02619580_g1).

Strains containing the empty YCplac33 plasmid or a plasmid containing a PWP2 allele were precultivated in selection medium (lacking uracil) and containing 2% galactose. The generation time was determined by measuring the increase in cell density upon cultivation in 96-well plates at 30 °C or 37 °C in selection medium containing either galactose (gPWP2 “ON”) or glucose (gPWP2 “OFF”) using a Tecan (Infinite 500) reader. Alternatively, the generation time at 23 °C was determined by measuring the increase in cell density upon cultivation in 20-ml cultures in selection medium containing either galactose (gPWP2 “ON”) or glucose (gPWP2 “OFF”).