Spark multimode plate reader

Cells were inoculated in YPD and grown overnight. The following day, cells were resuspended in fresh media, YPD or YPL, and grown for ~6 h. Cells were then diluted to an OD₆₀₀ of 0.05 in fresh YPD or 0.1 in fresh YPL, and 100 μl of culture was pipetted into a 96 multiwell flat-bottom transparent plate. The plate was shaken and kept at 30°C while the absorbance was measured every 30 minutes for 18 hours by the SPARK multimode plate reader (TECAN) according to the manufacturer’s instructions.

Luciferase reactivation was performed as described [82 (link),83 (link)] with slight modifications and scaled down to a 40 μl reaction volume. Briefly, 80 nM firefly luciferase (Sigma Aldrich) was denatured at 42°C for 5 min in a buffer of 25 mM HEPES (pH 7.5), 50 mM KCl, 15 mM MgCl₂, and 2 mM DTT, then placed on ice. BSA (0.05 mg/ml), ATP (1 mM), and an ATP regeneration system (20 mM creatine phosphate and 0.06 mg/ml creatine kinase) were added to the reaction mix, followed by addition of chaperones, cochaperones, and/or ribosomes. DnaK, CbpA, and GrpE were used at concentrations of 2 μM, 0.4 μM, and 0.2 μM, respectively, and 70S ribosomes were used at a concentration of 0.5 μM.
Renaturation of luciferase was allowed to proceed at 30°C. At the indicated times, 5 μl aliquots were removed. Luciferase activity was determined with the addition of 120 μl of substrate containing 200 μM D-luciferin (Sigma Aldrich), 0.5 mM ATP, and 10 mM MgCl₂. Luminescence was measured in a Spark Multimode plate reader (TECAN) with an integration time of 10,000 msec. The luminescence value at t = 0 was subtracted from subsequent measurements to determine the amount of luminescent activity regained.

The kinetic analysis of LdGSTu1 was conducted by steady state with varied concentrations of substrates 1-chloro-2,4-dinitrobenzene (CDNB) from 0.05 to 3 mM and p-nitrophenyl acetate (PNA) from 0.2 to 3.2 mM, while holding the GSH concentration constant at 5 mM, and for varied concentrations of GSH at 0.125 to 5mM while holding CDNB at a constant concentration of 2 mM. The reaction buffer was 100 mM KPi (pH: 6.5). Reactions were carried out in 96-well Greiner Bio-One UV-Star^® microplates (Sigma-Aldrich, St. Louis, MO, USA). Assays were run on a Spark^® multi-mode plate reader (Tecan Austria GmbH, Untersbergstr, Austria) in the kinetic mode, for a continuous read assay for 3 min. Product concentrations were calculated by path-length corrected molar attenuation coefficient from Habig et al. [64 (link),65 ]. Kinetic parameters and plots were calculated and generated with GraphPad Prism (GraphPad, San Diego, CA, USA).

Quantitative measurements of receptor-mediated inositol 1-phosphate (IP₁) were performed by competitive immunoassay utilising the IP-One assay kit (CisBio, France). HEK293 cells were prepared as described above and then assayed as per the manufacturer’s protocol. Briefly, at the time of assay, the culture media was removed from cells, and then replaced with IP-One stimulation buffer (HEPES 10 mM, CaCl₂ 1 mM, MgCl₂ 0.5 mM, KCl 4.2 mM, NaCl 146 mM, glucose 5.5 mM, LiCl 50 mM, pH 7.4). Cells were allowed to equilibrate in the stimulation buffer at 37 °C for 15 min, followed by addition of peptide ligands (to final concentrations of 0.1 pM – 10 µM) for subsequent stimulation for 1 h at 37 °C. The stimulation was terminated by lysis and the simultaneous addition of homogenous time-resolved fluorescence resonance energy transfer reagents. The lysates were incubated for a minimum of 1 h at room temperature. Fluorescence emission measurements at 620 nm and 665 nm were performed using a FlexStation3 plate reader (Molecular Devices, Sunnyvale, CA) or Spark Multimode plate reader (Tecan, Männedorf, Switzerland) at an excitation wavelength of 340 nm. Results were analysed as a ratio of fluorescence intensities of 665 nm to 620 nm.

A Staphylococcus aureus isolate (SA2; NCTC 178582), a methicillin-resistant (RC1; NCTC 12493), and a methicillin-susceptible S. aureus strain (SC1; NCTC 12981) were obtained from the University of Surrey Strain Collection (Guildford, UK) and cultured aerobically on brain heart infusion (BHI) agar (Sigma Aldrich, Poole, UK) at 37 °C for 24 h. AVNP suspensions were serially diluted 2-fold in Mueller-Hinton broth (MHB; Sigma) (1–0.007%) and added to 96 well plates containing an equal volume of MHB and 1 × 10⁵ colony forming units (cfu) of S. aureus. Plates were incubated at 37 °C, aerobically in a shaking incubator at 150 rpm for 24 h. Following incubation, a 20 μL aliquot of each sample was plated out onto MH agar and incubated at 37 °C, aerobically for 24 h before assessment of bacterial growth. The minimum bactericidal concentration (MBC) was determined as the lowest broth dilution of AVNP, which prevented S. aureus growth on the agar plate. To assess the effects of AVNP concentration on S. aureus growth over time, suspensions of AVNP (diluted from 0.03%, 0.015% and 0.007% (w/v) to avoid impairments with spectrophotometry readings) and S. aureus were incubated as described above, and optical density (600 nm) measured after 0, 8, 16 and 24 h using a Spark multimode plate reader (Tecan, Männedorf, Germany).

The Alamar-blue assay was utilized where resazurin was converted to resorufin in metabolically active cells with the resulting elevation in absorbance being proportional to the levels of viable cells. Briefly, A375, A431, and HaCaT cells were seeded into 96-well plates in 100 µL/well and incubated overnight prior to exposure to PZDHA. Cell density of A375 was 8000 and 4000 cells/well and for A431 and HaCaT 10,000 and 5000 cells/well for 24 and 48 h, respectively. On the following day, cells were exposed to a range of concentrations (1, 10, 25, 50, 75, and 100 µM) over different incubation periods. For control conditions, cells were incubated with complete medium only or medium containing 0.1% DMSO (vehicle). At the indicated time points, fresh medium (containing 0.1 mg/mL resazurin) was added into each well and incubated for 2–4 h (depending on the type of cancer cell line), at 37 °C. The plates were then centrifuged, and absorbance was recorded at 570 nm and 600 nm (reference wavelength) using a Spark multimode plate reader (Tecan, Switzerland). The levels of cell viability were estimated and expressed as percentage of control cells.

Peroxidated lipids were quantified by the Lipid Peroxidation Assay kit following manufacturer’s protocol (Abcam: ab118970). Briefly, Min6 cells were exposed to substrate limited media and given 3 mM glucose or 16.7 mM glucose for 45 min followed by 1 μM oligomycin in the high glucose group. Cells were lysed and a fluorescent adduct was generated of malondialdehyde present in cell lysates and thiobarbituric acid that was detected fluorometrically by a Tecan Spark Multi-mode plate reader. Reactions were performed in technical duplicates.