The release of DEX was monitored as a function of incubation time in DI water at 37°C. The number of samples for each group was four (n=5). PPy MCs were fabricated on 1cm × 1.5cm Au electrodes according to the previously-described protocol of electrospraying and electrodeposition methods.[50 –52 (link)] DEX (25mg) was dissolved in 1000ml of DI water to create solutions with a concentration of 0.025mg ml−1. All samples were sterilized with UV light for 24hrs. In order to load the samples, 1ml of DEX solution was pipetted onto each substrate, and degassed using low vacuum (Welch model 2026). Samples were kept in an incubator (Galaxy 170S, New Brunswick) at 37°C for 24hr. This process was performed 3 times. The samples were then immersed in 4ml DI water. The concentration of released DEX was measured at specific times using UV-Vis spectrophotometry (Molecular Devices SpectaMax M5) at 242nm wavelength.
Spectamax m5
Manufactured by Molecular Devices
Sourced in United States
The SpectaMax M5 is a multi-mode microplate reader that can measure absorbance, fluorescence, and luminescence in a wide range of microplate formats. It is designed to provide accurate and reliable data for various applications in life science research.
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Lab products found in correlation
Human il 18 platinum elisa, by Thermo Fisher Scientific (1 mentions)
Human tnfα elisa ready set go, by Thermo Fisher Scientific (1 mentions)
Human il 1β elisa, by Thermo Fisher Scientific (1 mentions)
N8010560, by Thermo Fisher Scientific (1 mentions)
Sytox green, by Thermo Fisher Scientific (1 mentions)
Cm h2dcfda, by Thermo Fisher Scientific (1 mentions)
Nitric oxide assay kit, by Abcam (1 mentions)
8 protocols using spectamax m5
1
DEX Release from PPy Microcarriers
2
Quantifying Cell Proliferation via MTT Assay
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The colorimetric MTT assay was modified and executed to quantify cell proliferation [25] (link). Exponentially growing MCF-7 and MCF-7/ADR cells were seeded in 96-well plates at a final concentration of 5×103 cells/well. After incubation for 24 h, cells in designated wells were treated with different concentrations of EVO. After 24, 48 and 72 h incubation, cell viability was detected by with the addition of free serum DMEM medium containing 1 mg/ml MTT for 4 h and subsequently dissolving the formed formazan crystals with DMSO. The absorbance in each individual well was determined at 570 nm by microplate reader (SpectaMax M5, Molecular Devices). The proliferation rates of cancer cells were evaluated by using triplicate assays. The LDH release rates from cells were evaluated by a commercial kit according to the manufacturers' protocol (Roche).
3
Quantification of Inflammatory Cytokines
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Human IL-18, IL-1β, and TNF-α were measured with the Human IL-18 Platinum ELISA, Human IL-1β ELISA and Human TNF-α ELISA Ready-SET-Go kits respectively (eBiosciences, San Diego CA) using 50 µL of plasma or 100 µL of culture supernatant. Each sample was tested in duplicate. Data was acquired using a SpectaMax M5 (Molecular Devices, Sunnyvale CA). The LLOD of IL-18 was 40 pg/mL in plasma and 18 pg/mL in culture supernatant.
4
Plasma Analyte Profiling Protocol
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Whole blood obtained from EDTA tubes were incubated overnight, and Ficoll density centrifugation was performed the next day to obtain plasma. Each plasma sample was subjected to a single free-thaw cycle and tested for 17 distinct analytes. IL-18 was measured using the human IL-18 ELISA kit (MBL). The assay was performed per the manufacturer's recommendations. In brief, all samples were diluted 1:5 in assay buffer and reported as pg/mL. Data were acquired using a SpectaMax M5 (Molecular Devices). The LLOQ of IL-18 in serum samples is 25 pg/mL. The Meso Scale Discovery (MSD) multiplex cytokine, proinflammatory and chemokine assays were used to assess 16 additional analytes: IL12/23p40, IL-15, IL-16, IL-7, IFN-g, IL-10, IL-1b, IL-2, IL-6, IL-8,TNF-a, Eotaxin, IP-10, MCP-1, MIP-1a, MIP-1b. The assay was performed per the manufacturer's recommendations. Data were acquired on a SECTOR Imager 2400. Results were analyzed using Meso Scale Discovery Workbench software. The LLOQ is for each analyte is indicated where data is shown.
5
Cytotoxicity Assay of PRI-724 on Cells
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Cells were seeded in a 96-well-plate (Fisher Scientific, N8010560) at a density of 2,000 cells per well and treated with PRI-724 at 0, 1, 10, 25, 50, or 100 μM for 24, 48 or 72 hours. 20 μl of 20 μM Sytox Green (Fisher Scientific, S7020) was added into each well. The plate was incubated in the incubator for 15 mins and read by plate reader SpectamaxM5 (Molecular devices, MV02017) at ex/em 485/530 with a 515 nm cut off for dead cell reading. 20 μl of 6% Triton X-100 (Boston BioProducts, P-925) was added to each well and the plate was incubated in the incubator for 45 mins and was read again for total cell reading. The value of total cell reading subtracted by the dead cell reading was considered as viable cell reading [27 (link)]. All experiments were performed with at least three independent replicates. P values < 0.05 were considered as significant and determined by Student’s t-tests.
6
Quantifying Intracellular Oxidative Stress
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Cells were incubated in the dark with 10 μM of 5-(and 6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2-DCFDA; Molecular Probes, US) for 15 min at 37 °C, and the fluorescence intensity was detected by a microplate reader (SpectaMax M5, Molecular Devices, US) using excitation at 492 nm and emission at 517 nm wavelengths.
7
Cytotoxicity Evaluation of F68-VES/MIT Micelles
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In vitro cytotoxicity of F68–VES/MIT micelles was determined using MTT assay. MCF-7 and MDA-MB-231 cells were seeded in 96-well plates at a density of 5,000 cells per well and cultured in 100 µL DMEM medium overnight. The cells were then treated with free MIT and F68–VES/MIT micelles at concentrations ranging from 0.25 µM to 4 µM for 24 hours and 48 hours, respectively. Untreated cells and cells treated with blank F68–VES micelles at equal volumes of F68-VES/MIT micelles were served as the control. After incubation, the medium was discarded and replaced by culture medium containing 1 mg/mL MTT and incubated for additional 4 hours before 100 µL DMSO was added to each well to allow the dissolution of formazan. The resulting absorbance, which represented the cell viability, was recorded by a microplate reader (SpectaMax M5; Molecular Devices LLC, Sunnyvale, CA, USA) at 570 nm.
8
Nitric Oxide Quantification Protocol
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NO was measured by a Nitric Oxide Assay Kit (Abcam, Cambridge, UK) following the manufacturer's instruction. Briefly, 75 μL of cell supernatants was mixed with 5 μL enzyme cofactor solution and 5 μL nitrate reductase. Following 2 h of incubation for converting nitrates to nitrites, 5 μL enhancer was added to each sample and incubated for 30 min. 5 μL DAN Probe was then added and incubated for 10 min. After that, 5 μL NaOH was added in the mixture for 10 min. The fluorescence intensity was detected by a microplate reader (SpectaMax M5, Molecular Devices, USA) using excitation at 360 nm and emission at 450 nm wavelengths.
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