All chemicals and solvents were of analytical grade and obtained from Merck, Germany. The reactions were monitored by thin-layer chromatography (TLC, E-Merck Kieselgel 60 F254). The column chromatography was carried out with the indicated solvents using silica gel (particle size 0.040–0.063 mm) from Merck (Germany). The microwave-assisted reactions were performed by the microwave synthesizer (CEM Discover, USA) with continuous stirring and controlled temperature. Melting points (mp, °C) of all compounds were determined in an open capillary using a Gallenkamp melting point apparatus without any correction. The infrared (IR) spectra were recorded using a Shimadzu FT-IR (IRAffinity-1S) spectrometer. An Agilent Technology LC-mass spectrometer with ESI ionization (1100 series LC/MSD Trap) was used to record the mass spectra (MS). A Bruker Avance 500 (1H, 500 MHz; 13C, 125 MHz) NMR spectrometer was used to record the 1H NMR and 13C NMR spectra at ambient temperature using DMSO-d6 as solvent. Chemical shifts are reported in parts per million (ppm) relative to the residual solvent peak as follows: DMSO-d6 = 2.50 ppm (1H NMR) and DMSO-d6 = 40.00 ppm (13C NMR). The Multiskan microplate reader was used to measure optical density (OD) at 570 nm.
Microplate reader
Manufactured by MultiSciences Biotech
Sourced in United States
A microplate reader is an analytical instrument used to measure and analyze the absorbance, fluorescence, or luminescence of samples in a microplate format. It serves as a core tool for various applications in life science research, drug discovery, and diagnostic testing.
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Lab products found in correlation
Kieselgel 60 f254, by Merck Group (2 mentions)
Avance 500, by Bruker (2 mentions)
Shark chondroitin sulfate, by Merck Group (2 mentions)
Ft ir iraffinity 1s spectrometer, by Shimadzu (1 mentions)
Cell light edu apollo567 in vitro kit, by RiboBio (1 mentions)
Cck 8, by Dojindo Laboratories (1 mentions)
1100 series lc msd trap, by Agilent Technologies (1 mentions)
Silica gel, by Merck Group (1 mentions)
13 protocols using microplate reader
1
Microwave-Assisted Organic Synthesis
2
Quantification of Sulfated Glycosaminoglycans in Bovine Cartilage Explants
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Bovine cartilage explants were pretreated in serum free culture medium in the presence or absence of 100 µM kartogenin for 48 hours, and treated with or without IL-1β (10 ng/mL). The medium samples were collected after 24 hours, and equal volumes were analyzed for sulfated glycosaminoglycans. Shark chondroitin sulfate (Sigma) dissolved at a concentration of 0 to 500 µg/mL in culture medium was used to produce a standard curve. Twenty microliters of media were added to a 96-well plate, followed by 180 µL of dimethylmethylene blue (DMMB) reagent. After 5 minutes of incubation, absorbance was determined at 525 nm with a Multiskan Ascent microplate reader. The sulfated glycosaminoglycans content was then calculated from the standard curve.26 (link),27 (link)
3
Evaluating Cell Proliferation by CCK8 and EdU
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Cell proliferation rate was determined by the CCK8 assay and Edu assay. MDA-MB-231 cells in the logarithmic growth phase were digested with 0.25% trypsin-EDTA, and single-cell suspension was collected by centrifugation (at 1000 rpm for 3 min). For the CCK8 assay, the suspended cells were seeded into 96-well plates at a density of 5000 cells in each well. There were six replicates for each sample. After treatment with dimethyl sulfoxide (DMSO) or 1 μM diABZI for 24 h, 48 h, and 72 h, 10 μl CCK-8 (Dojindo Laboratories) solution was added to each well and cultured for 2 h in an incubator at 37 °C with 5% CO2. The absorbance of each well was read at 450 nm with a Microplate Reader (Multiskan FC). For the EdU assay, 5 × 105 MDA-MB-231 cells were seeded into 12-well plates containing 1 ml DMEM. When the cell confluency reached 70%, the proliferation rate was determined with the Cell-Light EdU Apollo567 In Vitro Kit (RiboBio) according to the manufacturer's instructions. The images were taken with a fluorescence microscope (Olympus IX53).
4
Cytotoxicity of MIL-101(Fe) Nanoparticles
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Briefly, HepG2 cells (5 × 103 cells/well) were uniformly seeded into 96-well plates and cultured in an atmosphere of 37°C and 5% CO2 overnight. Then, HepG2 cells were incubated with fresh culture medium containing different concentrations of MIL-101(Fe) NPs (1.5625, 3.125, 6.25, 12.5, 50, 100, and 200 μg/mL). After 24 h, 20 μL of MTT (5 mg/mL) reagent was added into the wells and incubated for another 4 h. Nest, 100 μL DMSO was added into each well. Finally, a Multiskan Sky microplate reader was used to measure absorbance at 490 nm.
5
Quantification of PGE2 and Cortisol
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Levels of PGE2 in culture supernatants were determined using an ELISA kit (cat. no. KGE004B; R&D Systems, Inc.) in accordance with the manufacturer's instructions. Assays were based on the combined use of a monoclonal antibody against PGE2 and an alkaline phosphatase-conjugated polyclonal antibody; p-nitrophenyl phosphate substrate was added, and the absorbance at 405 nm was analyzed using a micro Multiskan plate reader. The limits of detection were 10 and 1.4 pg/ml for PGE2. Positive controls were used in each experiment.
Cortisol levels were also determined in culture supernatants using an ELISA kit (cat. no. KGE008B; R&D Systems, Inc.), in accordance with manufacturer's instructions to indicate the conversion rate of cortisone. The assay required microtiter plates coated with purified antibody and the standards or samples, anti-cortisol antibody and horseradish peroxidase-labeled avidin were subsequently added. Absorbance (optical density) was read using a Multiskan microplate reader at 450 nm, following a substrate 3,3′,5,5′-tetramethylbenzidine color reaction. Sample concentrations were calculated using a standard curve.
Cortisol levels were also determined in culture supernatants using an ELISA kit (cat. no. KGE008B; R&D Systems, Inc.), in accordance with manufacturer's instructions to indicate the conversion rate of cortisone. The assay required microtiter plates coated with purified antibody and the standards or samples, anti-cortisol antibody and horseradish peroxidase-labeled avidin were subsequently added. Absorbance (optical density) was read using a Multiskan microplate reader at 450 nm, following a substrate 3,3′,5,5′-tetramethylbenzidine color reaction. Sample concentrations were calculated using a standard curve.
6
Microwave-Assisted Organic Synthesis
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All reagents and chemicals
were obtained from the commercial supplier Merck (Germany) and used
without further purification. Reactions were monitored by thin-layer
chromatography (TLC) carried out on silica gel plates (E-Merck Kieselgel
60 F254) using UV light as the visualizing agent. Silica
gel (0.040–0.063 mm) from Merck (Germany) was used for column
chromatography.
The microwave-assisted synthesis was performed
by a microwave synthesizer (CEM Discover) with continuous stirring
and infrared temperature sensors. Melting points (mp, °C) were
determined in an open capillary using a Gallenkamp melting point apparatus
(Sanyo Gallenkamp, U.K.). A Shimadzu FTIR (IRAffinity-1S) spectrometer
was used to record the infrared (IR) spectra. Nuclear magnetic resonance
(1H NMR and 13C NMR) spectra were recorded on
a Bruker Avance 500 (1H, 500 MHz; 13C, 125 MHz)
NMR spectrometer at ambient temperature using CDCl3 and
DMSO-d6 as solvents. Chemical shifts are reported in parts
per million (ppm) relative to the residual solvent peak as following:
CDCl3 = 7.26 ppm (1H NMR), DMSO-d6 = 2.50 ppm (1H NMR), CDCl3 = 77.16 ppm (13C NMR), and DMSO-d6 = 40.00 ppm (13C NMR). A liquid chromatography machine (Agilent Technologies 1100
series LC/MSD Trap) was used to record the mass spectra (MS). Optical
density (OD) was measured at 570 nm on a Multiskan microplate reader.
were obtained from the commercial supplier Merck (Germany) and used
without further purification. Reactions were monitored by thin-layer
chromatography (TLC) carried out on silica gel plates (E-Merck Kieselgel
60 F254) using UV light as the visualizing agent. Silica
gel (0.040–0.063 mm) from Merck (Germany) was used for column
chromatography.
The microwave-assisted synthesis was performed
by a microwave synthesizer (CEM Discover) with continuous stirring
and infrared temperature sensors. Melting points (mp, °C) were
determined in an open capillary using a Gallenkamp melting point apparatus
(Sanyo Gallenkamp, U.K.). A Shimadzu FTIR (IRAffinity-1S) spectrometer
was used to record the infrared (IR) spectra. Nuclear magnetic resonance
(1H NMR and 13C NMR) spectra were recorded on
a Bruker Avance 500 (1H, 500 MHz; 13C, 125 MHz)
NMR spectrometer at ambient temperature using CDCl3 and
DMSO-d6 as solvents. Chemical shifts are reported in parts
per million (ppm) relative to the residual solvent peak as following:
CDCl3 = 7.26 ppm (1H NMR), DMSO-d6 = 2.50 ppm (1H NMR), CDCl3 = 77.16 ppm (13C NMR), and DMSO-d6 = 40.00 ppm (13C NMR). A liquid chromatography machine (Agilent Technologies 1100
series LC/MSD Trap) was used to record the mass spectra (MS). Optical
density (OD) was measured at 570 nm on a Multiskan microplate reader.
7
Cytotoxicity Assessment of Dextrans
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The cytotoxicity of the dextrans was evaluated following the mitochondrial reduction of the MTT. Briefly, the assay was conducted to investigate the cellular enzyme’s capacity of reducing the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan in RAW 264.7 macrophages with an active metabolism. The cells were incubated with the dextrans (50 mM) at 37 °C in the presence of DMEM for 24 h. After incubation, traces of dextrans were removed by washing the cells twice with 200 μL PBS and applying MTT (1 mg/mL) dissolved in 100 μL of fresh medium to determine the effects of the samples on cell viability. Cells were then incubated for 4 h at 37 °C, in 5% CO2. The MTT-formazan product dissolved in 100 μL of ethanol was estimated by measuring the absorbance at 570 nm in a Multiskan Ascent Microplate Reader. Inhibition of MTT reduction is presented as a percentage of cell proliferation under no treatment conditions. Absorbance was measured at 570 nm [43 (link)].
8
Drug Release Profiles of Nanoliposomal Formulations
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To examine the drug release profile of CDDP and DADS from nanoliposomes, 10 µL of free CDDP, free DADS, Lipo-CDDP, Lipo-DADS, and Lipo-CDDP/DADS were mixed with 990 µL of ethanol: water (70:30, v/v) separately and put into a dialysis bag with 50 mL PBS as sink medium. Subsequently, the buffer solutions around the dialysis bag were collected at different time intervals of 0, 0.5, 2, 4, 6, 8, 10, 12, 16, 18, 24, 28, 32, 36, 40, 44 and 48 h and replaced with the same volume of PBS simultaneously. The collected solutions were analyzed at 212 nm (CDDP) and 290 nm (DADS), respectively, using the Multiskan Go microplate reader, and the percentage of drug release was calculated using the following Equation (2):
9
Quantifying Histone-Antibody Interactions
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Serial dilution of purified recombinant histones (H2A, H2B, H3.1 or H4), the H2A–H2B dimer, the H3.1–H4 tetramer or mononucleosomes were adsorbed overnight at 4°C onto a microplate (Maxisorp Nunc), blocked for 3 h in PBS 10% skimmed milk and washed in 10 mM Tris-HCl pH 8, 150 mM NaCl and 0.1% Tween 20 (TBST). Primary staining was performed with a mixture of purified HA-tagged VHH (200 ng/ml) with an anti-HA monoclonal antibody (1 µg/ml) diluted in TBST 5% skimmed milk for 1 h. After three washes with TBST, horseradish peroxidase (HRP)-conjugated secondary anti-mouse-IgG antibody (diluted 1:5000 in TBST 5% skimmed milk) was added for 45 min, washed before HRP was revealed with 50 µl of 3,3′,5,5′-tetramethylbenzidine chromogenic substrate (Sigma-Aldrich) added to each well. The reaction was stopped by adding 50 µl of 0.5 M H2SO4, and the OD450 was measured using a microplate reader (Multiskan). Three independent experiments were performed (n=3) and each point was performed in triplicate. Mean and standard deviation values were calculated using Microsoft Excel software.
10
Measuring Sulfated Glycosaminoglycans in Cartilage
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