Soluble Gal1 was determined using an in-house Enzyme-Linked Immunosorbent Assay (ELISA) as described (34 (link)). In brief, high-binding 96-well microplates (Costar; Corning) were coated with capture Ab (2 μg/mL purified rabbit anti-human Gal1 polyclonal IgG) in 0.1 M sodium carbonate, pH 9.5. After incubation for 18 h at 4 °C, wells were rinsed three times with wash buffer (0.05% Tween-20 in PBS) and incubated for 1 h at room temperature with blocking solution (2% Bovine Serun Albumin (BSA) in Phosphate buffer saline (PBS)). Samples and standards (100 μL) were diluted in 1% BSA–Tween-20 and incubated for 18 h at 4 °C. Plates were then washed and incubated with 100 ng/mL biotinylated detection Ab (purified rabbit anti-human Gal1 polyclonal IgG) for 1 h. Plates were rinsed three times before adding 0.33 μg/mL HRP-labeled streptavidin (Sigma-Aldrich) for 30 min. After washing, 100 μL 3,3’,5,5’-Tetramethylbenzidine (TMB) solution (0.1 mg/mL tetramethylbenzidine and 0.06% H2O2 in citrate–phosphate buffer, pH 5.0) was added to plates. The reaction was stopped by adding 2N H2SO4. Optical densities were determined at 450 nm in a Multiskan MS Microplate Reader (Thermo Fisher Scientific). A standard curve ranging from 2.5 to 160 ng/mL human rGal1 was run in parallel.
Multiskan ms microplate reader
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Multiskan MS microplate reader is a compact and versatile instrument designed for absorbance-based measurements in microplates. It can accurately measure a wide range of absorbance values across multiple wavelengths, making it suitable for various applications in life science research and clinical laboratories.
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7 protocols using multiskan ms microplate reader
1
Quantifying Soluble Galectin-1 by ELISA
2
FGF2 Quantification in Cell Culture
3
Barberry Extract Cell Proliferation Assay
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Cells were treated with different concentrations of barberry extracts (0.001–1000 μg/ml). After 48 h of incubation, cell proliferation was measured based on the MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide) reduction assay [12 (link)]. When MTT reacts with mitochondrial dehydrogenase in living cells, the blue fromazan crystals will be formed. Procedure in brief: 20 μl of MTT (5 mg/ml in PBS) was added to 200 μl wells (in one tenth of the total volume) and incubated for 4 h at 37°C and 5% CO2. After incubation the medium was removed and the formazan blue crystals were dissolved by 100 μl of acidic isopropanol (0.04 M HCl in isopropanol). By using Multiskan MS microplate reader (Thermo lab systems, USA) at 540 nm each well was read. The result of the test was expressed as a stimulation index (SI), which is optical density at 540 nm (OD540) of the test samples/OD540 of negative control.
4
Cellular Viability Assay with miR-133b Modulation
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The EC cell lines were seeded in 96-well plates, at a density of 5 × 103 per well, then transfected with miR-133b mimics, miR-133b inhibitors, si-SUMO1 or their respective NCs. A total of 10 µl CCK-8 solution was added to each well, after the cells were cultured for 24, 48 and 72 h. After incubation at 37 °C for 2 h, the absorbance was detected at 450 nm using a Multiskan MS microplate reader (Thermo Fisher Scientific, Inc.).
5
Clove Extract Effects on Cell Proliferation
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After 48 hours of incubation with various concentrations of clove extracts, cell proliferation was measured based on the MTT [3-(4, 5-dimethylthiazole-2-yl)-2, 5-diphenyl tetrazolium bromide] reduction assay [14 (link)]. In brief, after incubation, 20 μL of MTT (5 mg/mL in PBS) were added to 200 μL wells (in one tenth of the total volume) and incubated for 4 hours at 37°C and 5% CO2. Then medium was removed and the formazan blue crystals, which formed by reacting MTT with mitochondrial dehydrogenase in the living cells, were dissolved by 100 μL of acidic isopropanol (0.04 M HCl in isopropanol). The plates were read using a Multiskan MS microplate reader (Thermo Scientific Vantaa, Finland) at the wavelength of 540 nm. The result of the test was expressed as a Stimulation Index (SI), which is OD540 of the group test samples/OD540 of each group negative control.
6
Paeonol Cytotoxicity Evaluation in Cells
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The cells were plated in triplicate in 96 well plates at a density of 5 × 103 cells in 0.1 mL and incubated overnight. Serial dilutions of Paeonol at doses ranging from 0–300μM were added and incubated for another 24, 48, or 72 hrs at 37°C. MTT (1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan, Sigma-Aldrich, St. Louis, MO, USA) was added to each well of the plates followed by incubation at 37°C for 4 hrs. Media were carefully aspired and 200μL of DMSO was added to each well. The plates were then incubated for 15 mins at 37°C, protected from light and measured at 570 nm by a Multiskan MS microplate reader (ThermoScientific, Massachusetts, USA). The growth curves of Paeonol-treated cells were plotted based on OD values obtained from the assay. Subsequently, Paeonol concentrations below the IC50 values after 48 hrs treatment (100 and 150μM, IC10 and IC20, respectively) were selected for further study. Each experiment was performed in triplicate and with at least 3 individual experiments carried out.
7
Cytotoxicity Evaluation via MTT Assay
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The cytotoxicity of the substances was analysed using the colorimetric MTT assay; the test protocol for cytotoxicity evaluation was adopted from elsewhere [26 (link)]. Briefly, different concentrations of the substances (10 μM–2 mM) were added and cells were incubated for 24 h, at 37 °C in humid air (98%) containing 5% CO2. Four hours prior to the end of the exposure period, the supernatant was removed, and MTT (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, Sigma-Aldrich, St. Louis, MO, USA, #M5655) solution in PBS (0.1 mg/mL, 100 μL/well) was added to the cells.
Upon the completion of the exposure period, the supernatant was removed, and a lysis solution containing 0.1% SDS (Sigma-Aldrich, #L3771) solution in DMSO (Sigma-Aldrich, #D8418) was added. Plates were shaken for 5 min, placed on a Multiskan MS Microplate Reader (Thermo Labsystems, Santa Rosa, CA, USA), and the absorbance was read colorimetrically at 492 nm. Each experiment was repeated three times, with four replications.
Upon the completion of the exposure period, the supernatant was removed, and a lysis solution containing 0.1% SDS (Sigma-Aldrich, #L3771) solution in DMSO (Sigma-Aldrich, #D8418) was added. Plates were shaken for 5 min, placed on a Multiskan MS Microplate Reader (Thermo Labsystems, Santa Rosa, CA, USA), and the absorbance was read colorimetrically at 492 nm. Each experiment was repeated three times, with four replications.
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