To perform pyocyanin pigment quantification assay, P. aeruginosa H103 cells untreated (grown in presence of 1% DMSO) and treated with 100, 50, 25, 12.5, 6.25, 3.12, 1.6 and 0.8 μg mL–1 of P. lentiscus L. fruit extracts (PLFE1-4) or ginkgolic acid-enriched fractions (PLFE1-(2-4)) were grown on 96-well microtiter plate for 24 h at 37°C on a rotary shaker (180 rpm). Then, supernatants samples were collected by centrifugation and extracted with chloroform. The chloroform layer (blue layer) was acidified by adding 0.5 M HCl. The absorbance of the HCl layer (pink layer) was recorded at 520 nm using the Spark 20 M multimode Microplate Reader controlled by SparkControlTM software Version 2.1 (Tecan Group Ltd.) and the data were normalized for bacterial cell density (OD580nm).
Sparkcontrol software
Manufactured by Tecan
Sourced in Switzerland
SparkControl software is a tool that enables the control and operation of Tecan's Spark multimode microplate reader. It provides a user-friendly interface for configuring and executing various microplate-based assays and experiments.
Automatically generated - may contain errors
Lab products found in correlation
D luciferin, by GoldBio (4 mentions)
Spark plate reader, by Tecan (4 mentions)
Spark 20m, by Tecan (2 mentions)
Spark 10m multimode microplate reader, by Tecan (1 mentions)
Te cool, by Tecan (1 mentions)
Black walled 96 well plates, by Thermo Fisher Scientific (1 mentions)
White walled 96 well plate, by Thermo Fisher Scientific (1 mentions)
384 well microplate, by Greiner (1 mentions)
Enzchek phosphate assay kit, by Thermo Fisher Scientific (1 mentions)
White walled 96 well plate, by Corning (1 mentions)
Ts 100 thermo shaker, by Biosan (1 mentions)
Spark 10m plate reader, by Tecan (1 mentions)
Spark cyto 400, by Tecan (1 mentions)
12 protocols using sparkcontrol software
1
Quantifying Pyocyanin in P. aeruginosa
2
Fluorescent Tracking of Tyrocidine Peptides
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A fluorescence method was developed tracking the naturally occurring fluorophores found in the tyrocidine structure (Trp at position 3 and/or 4 and Tyr at position 7). Optimisation of the method and standard curve used to calculate the amount of peptide bound can be found in the Supplementary Materials . All fluorescence results were background subtracted to remove any effect the materials had on the fluorescence signal.
The incubation step used for the creation of the antimicrobial materials, was assessed to determine the rate of association of the peptides to the base material. Fluorescence readings were collected at Ex290, and Em342 for two hours by using the Tecan Spark 10 M Multimode Microplate Reader which is controlled by the Spark ControlTM software, both provided by Tecan Group Ltd. (Mennedorf, Switzerland).
The incubation step used for the creation of the antimicrobial materials, was assessed to determine the rate of association of the peptides to the base material. Fluorescence readings were collected at Ex290, and Em342 for two hours by using the Tecan Spark 10 M Multimode Microplate Reader which is controlled by the Spark ControlTM software, both provided by Tecan Group Ltd. (Mennedorf, Switzerland).
3
Fluorescence Anisotropy Analysis of P. aeruginosa
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Fluorescence anisotropy analysis of P. aeruginosa cells grown to mid-log phase (A580 = 0.4) were performed as previously described61 (link) with few modifications. Cell pellets were washed two times (7500 × g, 5 min, 25 °C) in 10 mM MgSO4 and resuspended in the same wash solution to reach an A580 of 0.1. One µL of a 4 mM of1,6-diphenyl-1,3,5-hexatriene (DPH) stock solution (Sigma-Aldrich) in tetrahydrofuran was added to 1 mL aliquot of the resuspended cultures and incubated in the dark for 30 min at 37 °C to allow the probe to incorporate into the cytoplasmic membrane. Measurement of the fluorescence polarization was performed using the Spark 20 M multimode Microplate Reader, equipped with an active temperature regulation system (Te-CoolTM, Tecan Group Ltd., Männedorf, Switzerland). Excitation and emission wavelengths were set to 365 and 425 nm, respectively, and the anisotropy (r) was calculated according to Lakowicz62 . Three measurements were performed for each sample and data were recorded using SparkControlTM software (Version 2.1, Tecan Group Ltd., Männedorf, Switzerland). The relationship between fluorescence polarization and membrane fluidity is an inverse one, where increasing anisotropy values correspond to a more rigid membrane and vice versa. All values are reported as means of triplicate analyses for each experimental variable.
4
Quantifying T-cell Cytolytic Capacity
5
In Vitro T-Cell Cytotoxicity Assay
6
Cytolytic Capacity of CAR T Cells
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The cytolytic capacity of CAR-modified human T cells was assessed through a luciferase-killing assay. CAR T cells were co-cultured with 1×104 target cells, U937-CD33high or OCiAML3-CD33low tumor cells, at various effector-to-target ratios in quadruplicate in white-walled 96-well plates (Thermo Scientific) in a total volume of 200 µL of cell media. Target cells were plated with non-signaling control CAR T cells (H195DEL) at the same cell densities to determine maximal luciferase expression as a reference (max signal). Twenty-four hours or 96 hours later, 15 µg D-Luciferin (Gold Biotechnology) dissolved in 50 µL PBS was added to each well. Emitted luminescence of each sample (sample signal) was detected in a Spark plate reader (Tecan) and measured using SparkControl software (Tecan). Percent lysis was determined as: 100 − [(sample signal/average max signal)×100].
7
Condensin II ATPase Assay with λ DNA
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ATPase assays were performed using the EnzChek Phosphate Assay Kit (Invitrogen). 50 μL reactions contained 200 nM recombinant condensin II in reaction buffer (40 mM Tris [pH 7.5] 15 mM NaCl, 10 mM KCl, 7.5 mM MgCl2, and 2.5 mM ATP), supplemented with 1 × MESG substrate and 1 × purine nucleoside phosphorylase, with or without 1.59 nM (50 μg/μL) bacteriophage λ DNA (48,502 bp; TaKaRa). The ratio between condensin II and λ DNA was 1 condensin II: 385 bp of DNA. Reactions were placed at 37°C on a 384-well microplate (Greiner Bio-One), and analyzed by spark multimode microplate reader with SparkControl software (Tecan). The initial ATPase rate from timepoint 2.5 min to 7.5 min was determined as ATP molecules hydrolyzed per condensin II complex per min.
8
Competitive Growth Kinetics Evaluation
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Overnight cultures of the relevant strains (Competitor = EcM-Gm-CFP, Engineered strain = E. coli JW3910: p63_AF043 + pMPES_AF01) were grown, from single colonies picked from selective LB agar plates, in 5 mL of supplemented M9 media (0.4% glycerol, 0.2 % casamino acids) containing all necessary antibiotics. The overnight cultures were diluted 1:1000 into fresh M9 media containing 10 μg/mL Gentamicin and relevant inducers and grown for 6 h. The cultures were diluted to an OD (700 nm) of 0.1 using fresh M9 media containing 10 μg/mL Gentamicin and relevant inducers. The cultures were then mixed at the specified ratio. 10 μL of the culture was inoculated into 115 μL of media containing Gentamicin and relevant inducers in the wells of a 96 well microtitre plate and the plate was covered with a Breathe-Easy sealing membrane. The plate was placed in a plate reader (Tecan Spark) and grown for shown number of hours at 37 °C with continuous double orbital shaking (2 mm, 150 rpm). Measurements of absorbance (600 nm and 700 nm) and fluorescence (CFP: ex = 430 nm, em = 490 nm, GFP: ex = 485 nm, em = 530 nm, mCherry: ex = 575 nm, em = 620 nm) were taken every 20 min using Tecan Spark Control software. Data were processed using FlopR40 (link) to produce population and subpopulation curves, and plotted using R60 with ggplot261 and dplyr62 (link).
9
Cytotoxicity Assay for NKG2C-expressing CD8+ T Cells
10
Proline Content Determination by Ninhydrin Assay
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Proline content was determined according to Carillo and Gibon [56 ]. Proline was extracted from the 0.1 g frozen tissue powder in 40% ethanol overnight at 4 °C. After cold extraction, the homogenate was centrifuged for 5 min at 14,000× g. An aliquot of extract (0.05 mL) was incubated with 0.1 mL of a ninhydrin reagent (1% (w/v) ninhydrin in 60% (v/v) acetic acid and 20% ethanol (v/v)) at 95 °C for 20 min on a TS-100 Thermo-Shaker (Biosan, Riga, Latvia). After cooling and brief centrifugation, an 0.1 mL aliquot of the reaction mixture was transferred to a 96-well microplate, and the absorbance was measured at 520 nm and 25 °C using Spark multimode microplate reader with SparkControl software (Tecan, Männedorf, Switzerland). Proline content was determined using a standard curve with proline, and the results were expressed in nmol/mg FW.
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