Measurements were carried out on several microplate readers (Tecan SAFIRE, Tecan, Switzerland; LB 943 Mithras2, Berthold, Germany; Synergy H1, BioTek, United States) at 25 °C. UV–vis spectra were recorded using a 96-well plate (transparent, polystyrene, flat bottom, Sarstedt, Germany) as sample holder with clear viewseal sealer (Greiner Bio-One, Austria). The spectra were obtained in the wavelength range from 340 nm to 500 nm or 800 nm for the hydrogels that release 4-nitrophenol or 5,5′dibromo-4,4′-dichloro-indigo, respectively. A 1 nm wavelength step size was using for the Tecan SAFIRE for enzyme detection and the LB 943 Mithras2 performing the E. coli DH5α strain detection, and a 2 nm was used for the Synergy H1 for the EHEC strain detection, respectively.
Safire
Manufactured by Tecan
Sourced in Switzerland, Austria, United States
The Safire is a multi-mode microplate reader developed by Tecan. It is designed to perform a wide range of absorbance, fluorescence, and luminescence-based assays in microplates. The Safire provides accurate and reliable measurements, with the ability to detect a variety of analytes and support various applications in research and diagnostic settings.
Automatically generated - may contain errors
Lab products found in correlation
Clear viewseal sealer, by Greiner (3 mentions)
Microplate reader, by Tecan (3 mentions)
Lb 943 mithras2, by Tecan (2 mentions)
96 well flat bottom black plate, by Greiner (2 mentions)
Celltiter glo luminescent cell viability assay, by Promega (2 mentions)
Apotome, by Zeiss (2 mentions)
Mg132, by Merck Group (1 mentions)
Nunclon delta surface, by Thermo Fisher Scientific (1 mentions)
Synergy h1, by Agilent Technologies (1 mentions)
Synergy h1, by Tecan (1 mentions)
Cell counting kit 8 reagent, by Dojindo Laboratories (1 mentions)
Pcdna3, by Tiangen Biotech (1 mentions)
Mtt solution, by Merck Group (1 mentions)
Cellcarrier 384 ultra, by PerkinElmer (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Spectramax m5, by Tecan (1 mentions)
Pierce lal chromogenic endotoxin quantitation kit, by Thermo Fisher Scientific (1 mentions)
Celltiter glo kit, by Promega (1 mentions)
Human osteoclast precursors, by Lonza (1 mentions)
Toyocamycin, by Bio-Techne (1 mentions)
53 protocols using safire
1
Microplate-based Spectroscopic Analysis
2
Fluorescence Intensity Measurements
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Measurements were carried out either with a Varian Cary Eclipse spectrometer (Mulgrave, Victoria, Australia) or with microplate readers (Tecan SAFIRE, Tecan, Switzerland or LB 943 Mithras2, Berthold, Germany) at 25 °C. Fluorescence spectra obtained with the Varian Cary Eclipse spectrometer were measured at a scan rate of 120 nm/min and a resolution of 5 nm for the excitation and emission, using a 1 mm path-length quartz cell (SUPRASIL, Hellma Analytics, Germany). Fluorescence intensity measurements, which were recorded on the microplate reader, were performed using 96-well plates (black, polystyrene, flat bottom, Sarstedt, Germany) as sample holder with clear viewseal sealer (Greiner Bio-One, Austria). A bandwidth of 12 nm was applied for both excitation and emission. The gain parameter was manually set to 70 for the Tecan SAFIRE and to 255 for the LB 943 Mithras2, respectively.
3
Proteasome Activity Quantification Protocol
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For quantification of the proteasome activity, the standard protocol of Bowen T. S. et al. was used [40 (link)]. Frozen tissue samples were homogenized in proteasome lysis buffer (10 mM Tris, 1 mM EDTA, 2 mM ATP, 20% glycerin, 4 mM dithiothreitol), sonicated, and centrifuged at 16,000 rcf at 4 °C for 10 min. To ensure that only the proteasomal activity was measured, the samples were treated with the proteasomal inhibitor MG132 (Sigma) in parallel as a negative control. Chymotrypsin-like, trypsin-like, and peptidylglutamyl-peptide hydrolyzing activities of the proteasome were analyzed using the fluorogenic peptides Suc-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (LLVY-AMC) (Sigma), benzonyl-Val-Gly-Arg-4-methylcoumarin (Bz-VGR-AMC) (Enzo Life Sciences, Exeter, UK), and benzyloxy-carbonyl-Leu-Leu-Glu-methylcoumarylamide (Z-LLE-AMC) (Sigma). Protein (20 µg) was incubated with reaction buffer (0.05 mol/L Tris-HCl, pH 8.0, 0.5 mmol/L EDTA) and 40 µmol/L of one of three fluorogenic peptides. The reaction was inhibited by addition of the proteasome inhibitor MG132. The kinetic fluorescence was detected by a spectrophotometer (Tecansafire, Tecan, Crailsheim, Germany) at 380 nm excitatory (Ex) and 440 nm emission (Em) wavelengths. The enzymatic activity was calculated from a calibration curve of free 7-amino-4-methylcoumarine (AMC) (Sigma).
4
Cathepsin X Activity Assay
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For evaluating cathepsin X activity, cell lysates were diluted to 0.125 mg/mL protein concentration in 100 mM acetate buffer pH 5.5 [15 mM acetic acid, 84.8 mM Na-acetate, 0.1% PEG 8000] with 5 mM L-cysteine and 1.5 mM EDTA. 50 uL of lysates were pipetted in duplicates to the wells of the black microtiter plate (Nunclon Delta Surface; Thermo Fisher Scientific, Waltham, MA, USA) and incubated at 37 °C for 10 min. Immediately, 50 µL of fluorogenic substrate Abz-FEK(Dnp)-OH [56 (link)] at 5.9 µM final concentration was added and its degradation was monitored continuously at 320 ± 5 nm excitation and 420 ± 5 nm emission with a spectrophotometer Tecan Safire (Tecan, Männedorf, Switzerland). Kinetic measurements were analysed by Magellan™ [57 ] software and data, expressed in RFU/sec, were normalised to the control sample at 0 h.
5
Cytotoxicity of CCM-Hb-NPs in Breast Cancer
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The cytotoxicity of CCM-Hb-NPs against human breast cancer cell line MCF-7 was measured by Counting Kit-8 (CCK-8) assay. CCK-8 was used to determine the living cells. Briefly, the cells were seeded in 96-well plates (5 × 103 cells per well in 100 μL media) and cultured overnight. The culture medium was replaced with a fresh one containing equivalent doses of free CCM and CCM-Hb-NPs of 2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM and 60 μM, respectively. After incubation for 24 h, the culture medium was removed and cells were washed with PBS. Then 10% (v/v) CCK-8 solution in fresh medium was added and cells were incubated at 37 °C for 2 h. The absorbance of the solutions was then measured at 450 nm by a microplate reader (Safire; TECAN, Männedorf, Switzerland). Cells cultured in PBS were the control group. Inhibition Rate = (1-A value of CCM well/A value of control well) × 100%. Multiple groups were compared using one-way analysis of variance (ANOVA) followed by the Tukey-Kramer test for post hoc comparisons. Statistical significance was set at p < 0.05.
6
Measuring Hydrogel Bead Permeability
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The permeability
of the microbeads was studied by measuring the diffusion of N-dodecanoyl-l -homoserine lactone-3-hydrazone-fluorescein
(FITC-AHL) into the hydrogel beads. 0.3 mg of alginate-based beads
in 1 mL of CaCl2 Tris buffer (100 mM) was filtered using
cell strainers, and excess water was absorbed using filter paper before
they were immersed into 1 mL of FITC-AHL Tris solution (1.0 ×
10–5 mol/L) for 1, 2, 3, 5, or 10 min. The FITC-AHL
concentration of the supernatant was monitored. Beads were filtered
and immersed into 1 mL of 50 mM sodium citrate solution to release
FITC-AHL inside beads. The concentration of the released FITC-AHL
was determined using a microplate reader (Tecan SAFIRE, Tecan, Switzerland)
with a black flat-bottom 96-well plate (Greiner Bio-one, Germany).
Fluorescence intensity vs concentration standard curves of FITC-AHL
in Tris buffer and 50 mM sodium citrate solution were studied and
plotted.
of the microbeads was studied by measuring the diffusion of N-dodecanoyl-
(FITC-AHL) into the hydrogel beads. 0.3 mg of alginate-based beads
in 1 mL of CaCl2 Tris buffer (100 mM) was filtered using
cell strainers, and excess water was absorbed using filter paper before
they were immersed into 1 mL of FITC-AHL Tris solution (1.0 ×
10–5 mol/L) for 1, 2, 3, 5, or 10 min. The FITC-AHL
concentration of the supernatant was monitored. Beads were filtered
and immersed into 1 mL of 50 mM sodium citrate solution to release
FITC-AHL inside beads. The concentration of the released FITC-AHL
was determined using a microplate reader (Tecan SAFIRE, Tecan, Switzerland)
with a black flat-bottom 96-well plate (Greiner Bio-one, Germany).
Fluorescence intensity vs concentration standard curves of FITC-AHL
in Tris buffer and 50 mM sodium citrate solution were studied and
plotted.
7
Cell Viability Assay with CCK-8
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A total of 25 μl/well Cell Counting Kit-8 reagent (DOJINDO Inc., Japan) was added to each well of DSeA-3D plate after incubation of each tissue for 6 days at 37°C under 5% CO2. After incubation in 5% CO2 at 37°C for 2 hours, the absorbance at 450 nm in each well was measured using the microplate reader SAFIRE (TECAN Inc., JAPAN). The data subtracted from the value of blank well from each sample well were calculated.
8
UV-Vis Spectroscopy of Bacterial Cultures
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UV−Vis spectroscopy measurements were carried out on a microplate reader (Tecan SAFIRE, Tecan, Switzerland) at 25 °C. The absorbance of the cultured bacterial suspension at 600 nm was recorded using a 96-well plate (transparent, polystyrene, flat bottom, Sarstedt, Germany) as a sample holder with a clear viewseal sealer (Greiner Bio-One, Kremsmünster, Austria).
9
Cytotoxicity Evaluation of Nanoaggregates
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The cytotoxicity of the free drug, blank and loaded nanoaggregates was evaluated by MTT assay. Briefly, 100 l of MCF-7 cell suspension was transferred to a 96-well tissue culture plate (~10 4 cells per well or ~10 5 cells/ml) and allowed to attach for 24 h. After the cells were washed once with supplement-free medium, the sample was added and the cells were incubated for 24 h. Afterwards, the samples were removed, the wells were washed twice with supplement-free medium and replaced with 100 l of medium. 25 l of MTT solution in PBS (5 mg/ml) were added to each well. After 4 h of incubation the medium was removed and the dye was dissolved in DMSO. Orbital shaking at 300 rpm for 15 min was used to completely solubilize the crystals and the absorbance was measured at = 570 nm in a microplate reader (Safire, Tecan AG, Salzburg, Austria). Relative viability values were calculated by dividing individual viabilities by the mean of the negative control (untreated cells). 4% Triton X-100 in PBS was used as a positive control.
10
Cytotoxicity Evaluation of Compounds
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The cytotoxicity of the compounds was determined using an MTT assay. Briefly, about 104 cells per well in 100 µL of medium were seeded in a 96-well tissue culture plate and allowed to attach for 24 h. Before the experiment, the cells were washed twice with supplement-free ECM before the addition of the sample. The cells were incubated for 3 h. Subsequently, the samples were removed and replaced with 100 µL of supplement-free ECM. An MTT solution in PBS with a concentration of 5 mg/mL of thiazolyl blue tetrazolium bromide was prepared, and 25 µL was added to each well. The medium was again removed after 4 h, and the dye was dissolved in DMSO. The plate was mixed with orbital shaking at 300 rpm for 15 min. The absorbance was measured at λ = 570 nm in a microplate reader (Safire, Tecan AG, Salzburg, Austria). Relative viability values were calculated by dividing the individual values by the mean of the control. We used 4% Triton X-100 in PBS as a positive control.
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