Cells were seeded on 96-well microplates (Nest Scientific, Rahway, NJ, USA) at a density of 4 × 105 (HepG2) and 3 × 105 (human fibroblasts) in 100 μl of medium per well. The next day, the medium was removed and replaced with fresh medium containing dilutions of Cur and diamond nanoparticles (at concentrations of 15, 25, 45 and 75 mg/L) and their complexes at concentrations of 60 and 100 mg/L (Cur15:DN45, Cur45:DN15, Cur25:DN75 and Cur75:DN25). Cell viability was assessed after 24 h by MTT assay, where yellow soluble tetrazolium salt is converted to purple formazan crystals. MTT was dissolved in PBS (5 mg/ml) and 15 μl were added per well. After 3 h, solubilisation detergent (10% SDS, 0.01 M HCl) was added (100 μl /well). Spectrophotometer readings were performed on the following day at 570 nm on an Infinite® 200 PRO microplate reader with i-control™ Software (Tecan Group Ltd., Männedorf, Germany). USA). Cell viability was expressed as the percentage of the control group viability, which was 100%. Calculations were performed from the following formula: ABStest- ABSblank) /(ABScontrol-ABSblank), where “ABStest” is the absorbance of wells exposed to the treatment, “ABScontrol” is the absorbance of control wells, and “ABSblank” is the absorbance of wells without cells, with media containing the respective treatment for each well.
I control software
Manufactured by Tecan
Sourced in Switzerland, Germany, United States, Austria
I-control software is a comprehensive control and data analysis software for Tecan laboratory instruments. It provides intuitive interfaces and advanced functionalities to manage instrument operations and data processing.
Automatically generated - may contain errors
Lab products found in correlation
Infinite 200 pro, by Tecan (15 mentions)
Infinite m200 pro, by Tecan (8 mentions)
Infinite m200 pro plate reader, by Tecan (6 mentions)
Infinite 200 pro microplate reader, by Tecan (4 mentions)
Infinite 200 pro plate reader, by Tecan (4 mentions)
Infnite 200 pro microplate reader, by Tecan (3 mentions)
Infinite m200 pro reader, by Tecan (3 mentions)
Infinite m1000 pro, by Tecan (3 mentions)
Infinite 200 pro spectrophotometer, by Tecan (2 mentions)
Bio plex pro human screening panel 5plx exp, by Bio-Rad (2 mentions)
Elisa max deluxe set, by BioLegend (2 mentions)
Infinite plate reader, by Tecan (2 mentions)
96 well white polystyrene microplate, by Corning (2 mentions)
Celltiter glo luminescent cell viability assay, by Promega (2 mentions)
Infinite m200 microplate reader, by Tecan (2 mentions)
Nanoquant plate, by Tecan (2 mentions)
Prl tk, by Promega (2 mentions)
Infinite m200 pro microplate reader, by Tecan (2 mentions)
Infinite m1000, by Tecan (2 mentions)
Infinite m200 plate reader, by Tecan (2 mentions)
91 protocols using i control software
1
Evaluating Curcumin-Diamond Nanoparticle Cytotoxicity
2
Quantitative Rat CRP Liver Analysis
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Quantitative measurement of rat C-reactive protein (CRP) present in liver tissue was performed using an Enzyme-Linked Immunosorbent Assay kit (Abcam®). The provided CRP standard is used to prepare a standard curve to calculate the direct amount of the protein in samples. The minimum detectable amount of the protein for this assay is 0.7 ng/ml.
The homogenate was prepared as described above, in diluting buffer provided by the manufacturer (50 mg of liver tissue per 500 μl of buffer). The most suitable dilution of supernatant was determined empirically by performing a series of dilutions of pooled samples, in the range from 2 to 20 000. The 1:2000 dilution was chosen for this analysis since the detectable absorbance was within reader’s lower and upper limits. The assay was performed in accordance with the manufacturer’s protocol. Absorbance measurements were performed employing an Infinite® 200 PRO microplate reader with i-control™ Software (Tecan Group Ltd, Männedorf, Switzerland). Each sample was analyzed in duplicate.
The homogenate was prepared as described above, in diluting buffer provided by the manufacturer (50 mg of liver tissue per 500 μl of buffer). The most suitable dilution of supernatant was determined empirically by performing a series of dilutions of pooled samples, in the range from 2 to 20 000. The 1:2000 dilution was chosen for this analysis since the detectable absorbance was within reader’s lower and upper limits. The assay was performed in accordance with the manufacturer’s protocol. Absorbance measurements were performed employing an Infinite® 200 PRO microplate reader with i-control™ Software (Tecan Group Ltd, Männedorf, Switzerland). Each sample was analyzed in duplicate.
3
Quantification of TNF and Ach in Mice, Porcine, and Human Samples
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For mice, TNF levels were measured by ELISA (Mouse TNF-alpha DuoSet, R&D Systems) following manufacturer instructions and normalized to sham stimulated animals. The limit of detection was 15 pg./ml. Porcine samples were analysed by ELISA for quantification of TNF using Porcine DuoSet ELISA kit (Bio-Techne Ltd., cat. no. DY690B). Plates were analysed using the Infinite® 200 PRO spectrophotometer and iControl software (Tecan Group Ltd.).
Human samples were analysed for concentration of TNF using TNF alpha Human ELISA Kit (Invitrogen, cat. no. KHC3011) and for Ach using the Universal Acetylcholine ELISA Kit (Colorimetric) (Bio-Techne Ltd., cat. no. NBP2-66389) according to manufacturer’s instructions. Plates were analysed using the BMG FLUOstar Optima plate reader (BMG Labtech).
Human samples were analysed for concentration of TNF using TNF alpha Human ELISA Kit (Invitrogen, cat. no. KHC3011) and for Ach using the Universal Acetylcholine ELISA Kit (Colorimetric) (Bio-Techne Ltd., cat. no. NBP2-66389) according to manufacturer’s instructions. Plates were analysed using the BMG FLUOstar Optima plate reader (BMG Labtech).
4
Quantification of Hepatic Lipid Peroxidation
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Quantification of lipid peroxidation in liver tissue was performed using a Lipid Peroxidation (MDA) Assay Kit (Abcam®). Lipid peroxidation forms malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) as natural byproducts. The MDA in the sample reacts with thiobarbituric acid (TBA) to generate an adduct, which can be quantified colorimetrically (λ = 532 nm or fluorometrically (Ex/Em = 532/553 nm). The minimum detectable amount of MDA is 1 nmol/well and 0.1 nmol/well using the colorimetric and fluorimetric methods, respectively.
The homogenate was prepared as described above, in lysis buffer containing butylated hydroxytoluene (BHT) provided by the manufacturer (10 mg of liver tissue per 300 μl of MDA Lysis Buffer). Further steps were performed in accordance with the manufacturer’s protocol, including an additional step of filtering samples through a 0.22 μm syringe filter to eliminate turbidity. We performed both the colorimetric and fluorometric assays to choose the most accurate one. The measurements were performed employing an Infinite® 200 PRO microplate reader with i-control™ Software (Tecan Group Ltd.).
The homogenate was prepared as described above, in lysis buffer containing butylated hydroxytoluene (BHT) provided by the manufacturer (10 mg of liver tissue per 300 μl of MDA Lysis Buffer). Further steps were performed in accordance with the manufacturer’s protocol, including an additional step of filtering samples through a 0.22 μm syringe filter to eliminate turbidity. We performed both the colorimetric and fluorometric assays to choose the most accurate one. The measurements were performed employing an Infinite® 200 PRO microplate reader with i-control™ Software (Tecan Group Ltd.).
5
Splenic Nerve Stimulation and Noradrenaline Measurement
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Animal handling, management, anesthesia protocol, and surgical procedure were as above. In addition, after cuff implantation, a catheter was inserted into the SpV and routed toward the base of the spleen. Animals were then allowed to stabilize for 30 min. Blood (5 mL) was collected simultaneously from the SpV and the JV over 60 s. Splenic NVB stimulation (Stim 1) was performed 10 min later for 1 min at 10 Hz (bipolar, symmetrical biphasic rectangular pulses), and blood was collected as before. After 30 min, blood sampling was performed again during another baseline (Baseline 2) and stimulation (Stim 2) procedure. A sham (no current applied) stimulation was used as control. Samples were transferred immediately to ethylenediaminetetraacetic acid (EDTA) vacutainers, mixed by inversion, and stored on ice. Plasma was isolated by centrifugation (2,000 × g for 5 min), added to stabilizing solutions (as per enzyme-linked immunosorbent assay [ELISA] instructions), and immediately frozen on dry ice. Frozen plasma aliquots were thawed and immediately analyzed by ELISA for quantification of NA using the Noradrenaline Sensitive ELISA (DLD Diagnostika, category no. ea633/96) according to manufacturer’s instructions. Plates were analyzed using the Infinite 200 PRO spectrophotometer and iControl software (Tecan Group Ltd.).
6
Cytokine Quantification in Serum
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Serum concentrations of the cytokines interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-10, IL-17A and interferon (IFN)-γ were quantified using the Bio-Plex Pro Human Screening Panel 5plx EXP (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. For assay analysis, a Luminex® 200 device and Luminex XPonent® software version 3.1 (Luminex, Austin, TX, USA) were used. Tumor necrosis factor (TNF)-α was quantified using ELISA MAX™ Deluxe Sets (BioLegend) according to the recommended protocols of the manufacturer and the Tecan reader Infinite PRO 200 and the i-control™ software (both Tecan Group AG, Männedorf, Switzerland).
7
Cell Viability Measurement by MTT Assay
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Cell viability status was measured with the colorimetric MTT assay kit (ab211091, Abcam, Cambridge, MA, USA), according to the manufacturer’s protocol. Briefly, cells from the eighth-day chicken embryo femoral muscle were seeded into 96-well plate (Nest Scientific, Rahway, NJ, USA) at a density of 15 × 103 in 100 μL of medium per well in eight replicates per each group treatment. Then, cells were incubated with experimental factors (L-Glu, nGO) for 48 h, in standard conditions (at 37 °C in a humidified atmosphere containing 5% CO2). After that time, the growth medium was removed from the wells and cells were incubated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent for 3 h at 37 °C. After incubation, formazan crystals were dissolved in MTT solvent and incubated for 15 min. The absorbance of samples (n = 6) was measured at 570 nm on a microplate reader, Infinite® 200 PRO microplate reader with i-control™ software (Tecan Group Ltd., Männedorf, Germany). Calculations were performed as described by Strojny et al. [53 (link)].
8
Multiplex Cytokine Quantification
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The cytokines interleukin (IL)-2, IL-4, IL-10 and interferon (IFN)-γ were quantified in the serum samples using the Bio-Plex Pro Human Screening Panel 5plx EXP (Bio-Rad, Hercules, CA, USA), according to the manufacturer’s instructions. For multiplex assay analysis, a Luminex® 200 device and Luminex XPonent® software version 3.1 (Luminex, Austin, TX, USA) were used. The cytokines tumor necrosis factor (TNF)-α, IL-6 and IL-1β were quantified using ELISA MAX™ Deluxe Sets (BioLegend) according to the recommended protocols of the manufacturer and the Tecan reader Infinite PRO 200 and the i-control™ software (both Tecan Group AG, Männedorf, Switzerland).
9
Quantifying Oxidative Stress in EpiDerm
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The level of oxidative stress indicator in EpiDerm™ was measured 24 h after treatment with 8-hydroxy-2’-deoxyguanosine (8-OHdG) by an enzyme-linked immunosorbent assay (ELISA) kit (no. ab201734; Abcam, Cambridge, UK), following the instructions of the manufacturer. Absorbance values were measured on a microplate reader at 450 nm (Infnite® 200 PRO microplate reader with i-control™ software (Tecan Group Ltd., Männedorf, Germany)). The 8-OHdG concentration was calculated from the standard curve.
10
IL-1α Quantification by ELISA
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The medium for interleukin-1α (IL-1α) analysis was collected 24 h after the treatment (at day 2nd) and frozen at −80 °C. The concentration of this proinflammatory cytokine was detected with a colorimetric Human IL-1α ELISA Kit (Abcam, Cambridge, MA, USA) by strictly following the assay kit’s instructions. The measurement of the absorbance of the HRP/TMB colored base signal at 450 nm, from standards control and samples, was detected using an Infnite® 200 PRO microplate reader with I-control™ software (Tecan Group Ltd., Männedorf, Germany). Calculations were performed as described in the manufacturer’s protocol.
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