Purified biosensors were mixed with drug solutions to yield the mixture of 100 nM biosensor and the drug with desired concentrations. Samples were tested in at least three wells (see individual figures below). Solutions were in 3×PBS, pH 7.0 or 7.4. A Tecan Spark 10M plate reader was used to read the plate with 485 nm excitation and 535 nm emission wavelengths to measure GFP fluorescence (F0 and ΔF). The resulting data were fitted with Origin 9.2 software (OriginLabs) to the Hill equation [1 (link), 6 ].
Spark 10m plate
Manufactured by Tecan
The Spark 10M plate is a multi-mode microplate reader from Tecan. It is designed to perform absorbance, fluorescence, and luminescence measurements in 6- to 384-well microplates. The Spark 10M provides reliable and accurate results for a wide range of applications.
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4 protocols using spark 10m plate
1
Biosensors Characterization in Drug Solutions
2
Apoplastic ROS Burst Assays in Plants
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The POX assay in Arabidopsis and N. benthamiana was performed as previously described (Mott et al. 2016) . In brief, leaf disks (5 mm in diameter) were obtained from young leaves by coring and incubated with 50 µl of 1× MS buffer alone or supplemented with different peptides in a 96-well plate. After a 20-h incubation, the leaf disks were removed and each well received 50 µl of a 1-mg/ml solution of 5-aminosalicylic acid (A79809; Sigma-Aldrich), pH 6.0, with 0.01% H 2 O 2 . The reaction was allowed to proceed for 3 min and stopped by the addition of 2 N NaOH prior to reading the optical density at 600 nm on a Tecan Spark 10M plate reader. The luminol assay was performed as previously described (Baker and Mock 2004) . In brief, leaf disks (5 mm in diameter) were obtained from young leaves by coring and incubated overnight in a 96-well plate with water. The following day, 200 µM luminol, horseradish POX at 20 µg/ml, and peptides were added to each well. Isoluminol (600 µM) was used to achieve a similar magnitude for the flg22-triggered apoplastic ROS burst. Luminescence was measured using the Tecan Spark 10M plate reader.
3
Biofilm and Planktonic Growth Assay
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Colonies of PA14, SED42 and SED43 were taken from Luria-Bertani (LB) agar plates (Melford) and grown overnight at 37 °C. Colonies were used to inoculate replicate 100 μl aliquots of ASM1 or ASM2, ±3 μM glucose, in a 96-well microplate with a peg lid (Innovotech), n = 4 per growth condition. Cultures were incubated at 37 °C for 24 h. Growth of planktonic fractions of the populations was assessed by briefly shaking the well plate then reading absorbance at 600 nm using a Tecan Spark 10M plate reader. Biofilm formation on pegs was quantified using a crystal violet assay [42 (link)]. Briefly, pegs were rinsed with 150 μl phosphate-buffered saline (PBS) in a fresh 96-well plate to remove loosely-adhering cells. The peg lid was then transferred to a further 96-well plate with 150 μl 0.1% (w/v) crystal violet (Vickers Laboratories) per well. It was incubated at room temperature for 15 min and the pegs rinsed twice in PBS as above, before drying in a laminar flow hood for 30 min. The pegs were transferred to another 96-well plate containing 150 μl 95% (v/v) ethanol per well to solubilise the crystal violet. Absorbance of the solubilised crystal violet was read at 590 nm in a Tecan Spark 10M. The relative allocation of bacterial cells to the biofilm per well was calculated by dividing A590nm of crystal violet by A600nm of corresponding planktonic subpopulation.
4
Assessing Nuclear Membrane Permeability
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Nuclear membrane permeability was assessed by the binding of the cell impermeable CellTox Green dye (Promega) to nuclear DNA. Calu-3 cells were seeded in black 96 well plates (Corning) at a density of 1x10 4 cells per well and culture for 48 hours prior to assaying. Surfactant solutions (0.05-5.0 mM) were prepared in HBSS/HEPES (25 mM, pH 7.4). HBSS/HEPES buffer was used as the negative control and 0.2% Triton X-100 in HBSS/HEPES applied as positive control to induce total cellular permeabilisation.
Treatments were applied with 1% CellTox Green dye and exposed to cells for 4 hours.
Fluorescence was then measured at λex/em 495/519 nm using a Tecan Spark 10M plate reader.
To visualise changes in nuclear permeability, CellTox green uptake was imaged using fluorescence microscopy. Calu-3 cells were seeded in 12 well plates (Corning) at a density of 1.2x10 5 cells per well and cultured for 48 hours. Treatments were prepared with 1% CellTox Green dye as described above and applied to cells for 4 hours. Cells were then washed twice with PBS and fixed with 4% PFA for 10 minutes at room temperature. Cellular imaging was then performed on an EVOS® FL colour imaging microscope at 20X magnification using the GFP (λex/em 470/525 nm) and brightfield channels, and images merged using Image J (v1.52f).
Treatments were applied with 1% CellTox Green dye and exposed to cells for 4 hours.
Fluorescence was then measured at λex/em 495/519 nm using a Tecan Spark 10M plate reader.
To visualise changes in nuclear permeability, CellTox green uptake was imaged using fluorescence microscopy. Calu-3 cells were seeded in 12 well plates (Corning) at a density of 1.2x10 5 cells per well and cultured for 48 hours. Treatments were prepared with 1% CellTox Green dye as described above and applied to cells for 4 hours. Cells were then washed twice with PBS and fixed with 4% PFA for 10 minutes at room temperature. Cellular imaging was then performed on an EVOS® FL colour imaging microscope at 20X magnification using the GFP (λex/em 470/525 nm) and brightfield channels, and images merged using Image J (v1.52f).
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