Catheter segments loaded with NIC (2, 5 and 10% (w/w)) and non-loaded, with a length of 1 cm were weighted using a microbalance (Sartorius, Germany). NIC is a hydrophobic drug, and therefore is poorly soluble in aqueous solutions such as PBS33 (link). To perform an accurate quantification of released NIC, we supplemented PBS with Tween 80 in order to increase the solubility of NIC in PBS while increasing the saturation concentration of the drug34 (link). Then, each catheter segment was placed in 1 mL of phosphate buffered saline (PBS; ThermoFisher, USA) with 2% of Tween 80 (ThermoFisher, USA) and incubated at 37 °C with an agitation of 120 rpm (n = 3 per group). The buffer solution was exchanged for the fresh solution at every time point (1, 3, 4, 6 and 24 h, daily on days 2–10, and at 13, 16, 20 and 27 days). The aliquots were stored at − 20 °C for later use. The concentration of NIC released at every time point was calculated by measuring the absorbance at 340 nm of 300 µL of each aliquot in the flat bottom 96 well microtiter plates (Greiner Bio-One, USA) with a multi-well plate reader (Synergy H1, BioTek, USA). A calibration curve was plotted for NIC to estimate the concentration of drug released from the catheter segments. This calibration curve ranged from 1 to 50 µg/mL with R2 equal to 0.9998.
Flat bottom 96 well microtiter plate
Manufactured by Greiner
Sourced in Germany, Austria
The Flat-bottom 96-well microtiter plate is a laboratory equipment used for various applications in scientific research. It consists of a rectangular array of 96 individual wells with a flat bottom design. The plate is typically made of polystyrene or other compatible materials, ensuring compatibility with a wide range of laboratory instruments and assays.
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Lab products found in correlation
Co2 incubator, by Thermo Fisher Scientific (3 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Elx808 microplate reader, by Agilent Technologies (2 mentions)
Microbalance, by Sartorius (1 mentions)
Tween 80, by Thermo Fisher Scientific (1 mentions)
Synergy h1, by Agilent Technologies (1 mentions)
Fura 2 am, by Biotium (1 mentions)
Flexstation 3, by Molecular Devices (1 mentions)
Bodipy 581 591 c11, by Thermo Fisher Scientific (1 mentions)
Softmax pro microplate data acquisition analysis software, by Molecular Devices (1 mentions)
Cellytic b plus kit, by Merck Group (1 mentions)
Novagen bca protein assay kit, by Merck Group (1 mentions)
Automated microplate reader, by Agilent Technologies (1 mentions)
Golgistop monensin, by BD (1 mentions)
Breath easy sealing membrane, by Merck Group (1 mentions)
Infinite m200 pro plate reader, by Tecan (1 mentions)
Pd spintrap g 25, by GE Healthcare (1 mentions)
Infinite m200 pro, by Tecan (1 mentions)
Image scanner, by Epson (1 mentions)
Flat bottom 96 well microtiter plates, by Corning (1 mentions)
15 protocols using flat bottom 96 well microtiter plate
1
In Vitro Release Kinetics of Nicotine from Catheter Segments
2
Antifungal Compound Screening Protocol
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Drug susceptibility testing of all compounds was carried out in flat bottom, 96-well microtiter plates (Greiner Bio One) using the broth microdilution protocol according to the Clinical and Laboratory Standards Institute M-27A method. Growth inhibition of all strains was evaluated in the presence of varying concentrations of drug and reported as a % inhibition of untreated cells as previously described6 (link),7 (link). Experiments were repeated twice. Inhibition was measured based on OD595 growth data with or without compound (negative control). Compound screens for activity were determined against a panel of Candida species (fluconazole resistant or susceptible), Aspergillus fumigatus, and Cryptococcus neoformans.
3
Intracellular Calcium Dynamics in TRPA1 Cells
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Experiments were performed using a variant of previously published method58 . Briefly, CHO-TRPA1 cells were plated at a density 20000 cells/well onto poly-D-lysine-coated flat bottom 96-well microtiter plates (Greiner Bio-One, Belgium) and left to attach overnight. For experiments cells were incubated with 2 µM Fura-2 AM (Biotium, Hayward, CA, USA), at 37 °C for 60 min. Loaded cells were washed, re-suspended in the assay buffer (HBSS, 2 mM CaCl2, 2.5 mM probenecid, 10 mM HEPES adjusted with NaOH to pH 7.4) and placed into the FlexStation 3 (Molecular Devices, Sunnyvale, USA) to monitor fluorescence before and after the addition of the compounds of interest. The intracellular Ca2+ responses were determined at 37 °C from fluorescence emissions at 510 nm elicited by alternating excitation at 340 and 380 nm.
4
Cellular Lipid Peroxidation Assay
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CHO-mTRPA1 cells were seeded into flat-bottom 96-well microtiter plates (Greiner Bio-One) and grown to confluency. Cells were treated with APs or H2O2 (300 μM) in PBS for 10 and 30 min at 37 °C in a cell culture incubator. The compounds were removed and BODIPY 581/591 C11 (Thermo Fisher Scientific, Eugene, OR, USA, 10 μm final concentration) was added. After 30 min in culture, cells were washed and resuspended in PBS and the fluorescence of C11 BODIPY was monitored with excitation/emission of 581/591 nm for the reduced dye, and excitation/emission of 488/510 nm for the oxidized dye using a Flexstation III Benchtop Multi-Mode Microplate Reader and the SoftMax Pro Microplate Data Acquisition & Analysis Software (Molecular Devices). The ratio of the emission fluorescence intensities at 591 nm to 510 nm gives the read-out for lipid peroxidation in cells.
5
Quantifying Xylanase Activity in Cells
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Xylanase activity was measured with EnzChek® Ultra Xylanase Assay Kit (Life Technologies) according to manufacturer’s instructions. Briefly, cells were grown for 21 hours, reaching similar densities, harvested and lysed with CelLytic™ B Plus Kit (Sigma-Aldrich). Cell lysate and supernatant from cultures were diluted and 50 μL of dilutions were mixed with 50 μL of xylanase substrate working solution in flat-bottom 96-well microtiter plate (Greiner Bio-One). They were incubated at room temperature for 40 min and the release of reaction products was measured with the ELx808™ Microplate Reader (BioTek) with the excitation at 360 nm and emission at 460 nm. Total protein content was measured with Novagen® BCA Protein Assay Kit (Merck) and xylanase activity was normalized to it.
6
Quantification of sfGFP Expression in Geobacillus
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The sfGFP [34 (link)] was used as a reporter to assess the expression levels. It was previously shown to be active in Geobacillus species [27 ]. For quantification of sfGFP expression driven by different promoters and RBS’s, Geobacillus strains carrying the respective constructs were grown overnight at 60°C in TMM with 0.05% yeast extract and 0.2% glucose. 2 μL of these cultures were inoculated into 100 μL of fresh pre-heated media in flat-bottom 96-well microtiter plate (Greiner Bio-One) and sealed airtight with VIEWSeal (In Vitro) to prevent water evaporation. Plates were incubated at 60°C and 200 rpm. Periodically fluorescence was measured with the ELx808™ Microplate Reader (BioTek) with the excitation at 485 nm and emission at 535 nm. Values at the middle of log phase were taken for analysis. Fluorescence was normalized to OD600 measured at the same time.
7
Cytokine-Induced IL-1β and IL-10 Quantification
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The supernatants of IL-1β and IL-10 (BD Bioscience Pharmingen, Franklin Lakes, NJ, USA) were evaluated by ELISA kits according to the manufacturer’s protocol. Briefly, the ability of cells to secrete IL-1β in response to cytokines was determined using a sandwich ELISA. A flat-bottom 96-well microtiter plate (Greiner Bio-One, Kempten, Germany) was coated with 100 μl/well of anti-human IL-1β mAb (2 mg/ml in a mixture of sodium carbonate and sodium bicarbonate, pH 9.5) overnight at 4 °C. The plate was subsequently washed with phosphate-buffered saline (PBS; pH 7.0) and 0.05% Tween-20, and blocked with 10% fetal calf serum (FCS). IL-1β standards (rHu IL-1β) were made in a solution consisting of PBS (pH 7.0) and 10% FCS using serial dilutions. Standards or supernatants (100 μl/well) were plated in triplicate and incubated at ambient temperature for 2 h. After three washes, 100 μl/well of biotinylated anti-human IL-1β mAb (100 ng/ml in PBS, pH 7.0, and 10% FCS) was added, followed by 100 μl/well of streptavidin–peroxidase conjugate. The chromogen substrate was used at 100 μl/well; after 30 min, 10% H2SO4 was added to stop the reaction. Absorbance was read at 450 nm on an automated microplate reader (Bio-Tek Instruments, Richmond, CA, USA). IL-10 was also determined by ELISA using a specific ELISA kit.
8
Evaluating NK Cell Cytotoxicity via CD107a
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Since the lysosomal-associated membrane protein-1 (LAMP-1 or CD107a) in NK cells is expressed during degranulation and correlates with NK cell-mediated tumor cell lysis [50 (link)], the expression of CD107a on NK cells was used to evaluate natural and antibody-mediated NK cell cytotoxicity. TKI-sensitized and unsensitized IGROV-1 and SKOV-3 cells were co-incubated with purified unstimulated NK cells (1:1 cell ratio) on a flat-bottom 96-well microtiter plate (Greiner Bio-One). Cetuximab was directly added at 1 µg/mL in ADCC experiments. NK cells were labelled with anti-CD107a-FITC or isotype mIgG1-FITC 1:20, after one hour incubation at 37 °C in 5% CO2, GolgiStop monensin (BD GolgiStop, BD Bioscience) was added at a dilution of 1:600. After a further 5 h incubation at 37 °C in 5% CO2, cells were resuspended in 200 µL PBS with azide and immediately analyzed by flow cytometry.
9
Growth Kinetics of S. bombicola Strains
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Specific growth rates for S. bombicola strains were determined by incubating 200 μL of cell culture in Lang medium in a flat bottom, 96-well microtiter plate (Greiner Bio-One) at 30 °C in a Tecan Infinite® m200 Pro plate reader (Tecan, Mannedorf, Switzerland) with an initial OD600 of 0.05, sealed off with a Breath-Easy® sealing membrane (Sigma-Aldrich). The OD600 was measured every 15 min for 40 h, while shaking in between measurements at 200 rpm (orbital mode, amplitude 2 mm). The maximum specific growth rate (μ) was calculated by fitting the Richards model in with the ‘curve_fit ‘function in the ‘SciPy’ module in Python 3.7 [65 (link)].
10
Xylem Sap Protein Quantification
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Xylem sap protein concentrations were determined with Pierce® Coomassie Plus kit (Thermo Fisher Scientific), which is based on the Bradford method (Bradford, 1976) (link). Fifty µl xylem sap and 50 µl Coomassie Plus were mixed in a flat bottom 96 well micro titer plate (Greiner AG, Kremsmünster, Austria) and incubated for 10 min at room temperature. During incubation, the plates were centrifuged for 5 min at 4000 x g to remove bubbles. Absorbance was measured at 595 nm in a plate reader (Infinite M200 Pro®, Tecan Group AG, Männedorf, Switzerland). A dilution series ranging from 1.25 µg ml -1 to 10 µg ml -1 bovine serum albumin (BSA, Merck KGaA) was used for calibration and processed together with the samples.
To test, if the protein assays were disturbed by low molecular weight compounds in the xylem sap, 0.5 ml aliquots of xylem sap were filtered through Sephadex columns (PD SpinTrap® G-25, GE Healthcare, Chicago, Il, USA) using Tris-buffer (50 mM Tris HCl (Roth), 1 mM MgCl (Merck KGaA), pH 8.1) for column equilibration and sample elution. The protein concentrations were measured as above in the filtered extracts. The mean protein concentration differences between filtered and non-filtered xylem sap samples were less than 20% and not significant. Therefore, we used xylem sap without pretreatment for protein determination.
To test, if the protein assays were disturbed by low molecular weight compounds in the xylem sap, 0.5 ml aliquots of xylem sap were filtered through Sephadex columns (PD SpinTrap® G-25, GE Healthcare, Chicago, Il, USA) using Tris-buffer (50 mM Tris HCl (Roth), 1 mM MgCl (Merck KGaA), pH 8.1) for column equilibration and sample elution. The protein concentrations were measured as above in the filtered extracts. The mean protein concentration differences between filtered and non-filtered xylem sap samples were less than 20% and not significant. Therefore, we used xylem sap without pretreatment for protein determination.
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