The influence of pH on the adhesion of OAW42 cells was determined in vitro by a colorimetric cell adhesion assay (The CytoSelect 48-Well Cell Adhesion Assay Kit, CBA-070, USA). The absorbance was determined with the NanoQuant Infinite M200 Pro microplate reader (Tecan Austria GmbH, Grödig, Austria). After titrating the pH of the cell culture medium and supplementing 50% of the samples with 10% human ascites, OAW42 cells were grown in the incubator for 12, respectively, 24 h. Cells were suspended in serum-free media, and the suspension was pipetted into 40 ECM protein-coated (Fibronectin, Collagen I, Collagen IV, Laminin I, Fibrinogen) wells and 8 Bovine serum albumin (BSA)-coated wells and then incubated in a cell culture incubator. After PBS-washing, cells were stained by Cell Stain Solution (The CytoSelect 48-Well Cell Adhesion Assay Kit, CBA-070, USA). Then, cells were washed with deionized water, the supernatant extracted, and cells were transferred to a 96-well plate. Optical density was then measured by NanoQuant Infinite M200 Pro microplate reader (Tecan Austria GmbH, Grödig, Austria) at a wavelength of 560 nm.
Infinite m200 pro nanoquant
Manufactured by Tecan
Sourced in Switzerland, Austria, France
The Infinite M200 Pro NanoQuant is a multimode microplate reader designed for quantification and characterization of nucleic acids and proteins. It features high-performance absorbance and fluorescence detection capabilities.
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Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Sulforhodamine b srb sodium salt solution, by Merck Group (3 mentions)
Alamar blue, by Thermo Fisher Scientific (3 mentions)
Thrombin, by Merck Group (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Tri reagent, by Merck Group (2 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Novaseq 6000, by Illumina (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (2 mentions)
Biomate 5, by Thermo Fisher Scientific (2 mentions)
Ribopure rna extraction kit, by Thermo Fisher Scientific (2 mentions)
Pkcζ specific pseudosubstrate inhibitor, by Thermo Fisher Scientific (2 mentions)
Qiamp dna stool mini kit, by Qiagen (2 mentions)
Platinum quantitative pcr supermix udg, by Thermo Fisher Scientific (1 mentions)
Tri reagent rna isolation reagent, by Merck Group (1 mentions)
Moloney murine leukemia virus reverse transcriptase, by Promega (1 mentions)
Brdu cell proliferation kit, by Roche (1 mentions)
Bms223hs, by Thermo Fisher Scientific (1 mentions)
109 protocols using infinite m200 pro nanoquant
1
Influence of pH on OAW42 Cell Adhesion
2
Sulforhodamine B Assay for Melanoma Cells
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Melanoma cells were seeded in 96-well plates (12.0 × 103 cells/well for each cell line). The day after, cells were treated with HPF at the concentration of 3 µM, followed by an incubation of 48 h. At the end of the treatment, cells were fixed by adding 25 µL/well of 50% (w/v) trichloroacetic acid into the culture medium. Plates were then incubated at 4 °C for 1 h, washed 4 times with deionized water (ddH2O), and allowed to dry at room temperature (RT). To perform staining, 50 µL/well of 0.04% (w/v) sulforhodamine B (SRB) sodium salt solution (Sigma-Aldrich, Milan, Italy) was added. After incubating for 1 h at RT, the plates were rinsed with 1% acetic acid and allowed to air dry. SRB stain was then solubilized using a 10 mM Tris-base solution at pH 10.5. Subsequently, the absorbance of the samples was measured at 540 nm using a TECAN NanoQuant Infinite M200 Pro plate reader (Tecan Group Ltd., Männedorf, Switzerland). Five to six replicates were performed for each condition or data point.
3
Sulforhodamine B Cytotoxicity Assay
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Melanoma MeWo and A375 cells or NHEM were seeded in 96-well plates (5.0 × 103 cells/well for MeWo cells and 2.9 × 103 cells/well for A375 or NHEM). After 24-h culture, the cells were treated with different concentrations of compounds 1 –7 . After 72-h treatment, the cells were fixed by adding 25 µL/well of 50% (w/v) TCA directly into the culture medium. The plates were incubated at 4 °C for 1 h, washed four times with ddH2O, and dried at RT. Staining was performed by adding 50 µL/well of 0.04% (w/v) sulforhodamine B (SRB) sodium salt solution (Sigma-Aldrich, Milan, Italy). After 1-h incubation at RT, the plates were rinsed with 1% HAc and air-dried. SRB was solubilized in a 10 mM Tris-base solution pH 10.5 and Abs492 measured in the plate reader TECAN NanoQuant Infinite M200 Pro (Tecan Group Ltd., Männedorf, Switzerland). Six replicates for each condition/data point were performed.
4
Cytotoxicity Evaluation of HPF
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A375, FO1, SK-Mel-28 and NHEM cells were seeded in 96-well plates (A375: 3.0 × 103 cells/well; FO1, SK-Mel-28, MeWo and NHEM: 6.0 × 103 cells/well). After 24 h, cells were treated with different concentrations of HPF and incubated for 24, 48 and 72 h. At the end of each treatment, cells were fixed by adding 25 µL/well of 50% (w/v) trichloroacetic acid directly into the culture medium. Plates were incubated at 4 °C for 1 h, washed 4 times with ddH2O and dried at room temperature (RT). Staining was performed by adding 50 µL/well of 0.04% (w/v) sulforhodamine B (SRB) sodium salt solution (Sigma-Aldrich, Milan, Italy). After 1 h incubation at RT, plates were rinsed with 1% acetic acid and air-dried. SRB was solubilized in 10 mM Tris base solution, pH 10.5 and Abs 540 nm, measured in the plate reader TECAN NanoQuant Infinite M200 Pro (Tecan Group Ltd., Männedorf, Switzerland). Six replicates for each condition/data point were performed.
5
3T3-L1 Cell Viability Assay
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3T3-L1 cells were seeded in 96-well plates at a density of 2 × 103 cells/well. Cells were treated for 48 h with a DMSO solution at the final concentration of 10 µM DCQA in the presence or absence of a cocktail of differentiation (MDI). Cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,3-diphenyl tetrazolium bromide (MTT) assay. Briefly, 4 h before the end of the treatment, MTT reagent (0.5 mg/mL, 10 µL) was added to each well and incubated until treatment was completed. The colored crystals of produced formazan were dissolved in 200 µL of isopropanol and the Abs500 was measured in the plate reader Tecan NanoQuant Infinite M200 Pro (Tecan Group Ltd., Männedorf, Switzerland). Four replicates for each condition were performed. Three independent experiments were performed for each condition.
6
HT22 Cell mRNA Extraction and qPCR Analysis
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mRNA was isolated from harvested HT22 cells using TriReagent® RNA Isolation Reagent (Sigma-Aldrich; Merck KGaA) according to the manufacturer's protocol. mRNA concentrations were measured using a Nano Quant infinite M200 Pro (Tecan Group, Ltd.). Moloney Murine Leukemia Virus Reverse Transcriptase was used to synthesize first strand cDNA according to the manufacturer's protocol (Promega Corporation).
For TaqMan™ qPCR (7500 Real-Time PCR system, Applied Biosystems; Thermo Fisher Scientific, Inc.), 0.5 µl each of the forward and reverse primers, and the probe (all purchased from Eurofins Genomics), 2.5 µl cDNA template and 12.5 µl Platinum™ Quantitative PCR SuperMix-UDG (Thermo Fisher Scientific, Inc.) were added per well in a 96-well plate and amplified for 40 cycles (annealing temperature, 60̊C) according to the manufacturer's protocol. The sequences of the primers used and the respective probes are presented inTable SI . qPCR data of mRNA expression levels were normalized using the 2-ΔΔCq method in Microsoft Excel 2010 (Microsoft Corporation) using GAPDH as the reference gene (37 (link)).
For TaqMan™ qPCR (7500 Real-Time PCR system, Applied Biosystems; Thermo Fisher Scientific, Inc.), 0.5 µl each of the forward and reverse primers, and the probe (all purchased from Eurofins Genomics), 2.5 µl cDNA template and 12.5 µl Platinum™ Quantitative PCR SuperMix-UDG (Thermo Fisher Scientific, Inc.) were added per well in a 96-well plate and amplified for 40 cycles (annealing temperature, 60̊C) according to the manufacturer's protocol. The sequences of the primers used and the respective probes are presented in
7
HPLC Purification of IgM Protein
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Positive IgM fractions were analyzed by HPLC using an equilibrated Atlantis Waters/250 mm × 10 mm (10 μm)/dC18 column (Atlantis Columns) in Prominence LC-20A-HPLC-04 equipment (Shimadzu, Japan). The sample was injected (20 μL) and eluted under isocratic conditions, acetonitrile/H2O 60:40. The column was operated at 2.0 mL/min flow rate, 27°C and 15 bar. The presence of proteins was observed at 280 nm. The purified IgM protein was quantified using a NanoQuant Infinite M200Pro spectrophotometer (Tecan Group Ltd.).
8
Colorimetric Assay for Cell Proliferation
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Cell proliferation was assessed with a colorimetric immunoassay based on the measurement of Br-deoxy-uridine (BrdU) incorporation during DNA synthesis. A375, FO1 and SK-Mel-28 cells were cultured in a 96-well plate as previously described, and after 24 h, incubated with or without HPF. After 48 h of treatment, the cells were incubated for 8 h with BrdU labelling solution (BrdU Cell Proliferation Kit, Roche, Merck, Milan, Italy), and then fixed with 200 μL/well of fix/denaturing solution for 30 min at RT. After washing 3 times, the peroxidase goat anti-mouse IgG conjugate was added, and the plate was incubated for 90 min at RT. Three washes were performed again, then 100 μL/well of TMB peroxidase substrate solution was added and plates were incubated at RT until color development was sufficient for photometric detection (5–30 min). Then, the absorbance of the samples was measured at Abs 370 nm (reference wavelength: approx. 492 nm) in a Tecan NanoQuant Infinite M200 Pro plate reader (Tecan Group Ltd., Männedorf, Switzerland). After many measurements at various time points (e.g., 5, 10 and 20 min), the reaction was stopped with 25 μL/well of 1 M H2SO4 and was measured in a plate reader at 450 nm (reference wavelength: 690 nm).
9
Quantifying Inflammatory Mediators in Periodontal Cells
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ELISA assays were used on hPDL and OCM.30 cells to quantify the production of three inflammatory mediators: a growth factor, vascular endothelial growth factor A (VEGF-A), and two inflammatory cytokines, tumor necrosis factor alpha (TNF-α) and interleukin 11 (IL-11), involved in inflammatory reaction periodontitis [24 (link),25 (link)]. These assays were made at 3 days following direct contact of each two-cell types with GX, GX-100, GX-200, Emd®100, and Emd®200. All cell lysates were subjected to ELISA for inflammatory mediators by applying ELISA kits: human (for hPDL) and mouse (for OCM.30) VEGF-A (KHG011, ThermoFisher Scientific), TNF-α (BMS223HS, ThermoFisher Scientific, France), and IL-11 (human EHIL11 and mouse EMIL11, ThermoFisher Scientific, France). Each ELISA was performed according to the manufacturer’s instruction. The experiment was performed in triplicate and repeated three times (n = 9); the data were compared to a standard curve. Absorption measurements were performed at 450 nm (Nanoquant infinite M200 pro, Tecan group Ltd., Lyon, France).
10
Biocompatibility Evaluation of ALD and HA-ALD
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The biocompatibility of the tested compounds was evaluated by means of a quantitative analysis according to the ISO 10993–5:2012 International Standard. For cell viability assays, Saos-2 osteoblasts and primary bovine chondrocytes were plated at a density of 1 × 104 cells per well in 96-well plates (Corning, USA). After 24 h of incubation under standard conditions, the cells were washed with PBS 1X (Life Technologies, Italy), and solutions of ALD and HA-ALD were added to reach a final conc. of 3, 6, 12, 25, 50 and 100 μM (four replicates were tested for each condition). The cells were incubated for 24 h, 3 days and 7 days, and the medium was then aspirated. The cells were washed again with 1X PBS, and 100 μL of complete medium containing 10% Alamar Blue (Life Technologies, Italy) was added. The cells were subsequently incubated for 4 h under standard culture conditions, and finally, the fluorescence was measured using a microplate reader (Nanoquant Infinite M200 Pro, Tecan Group Ltd, Switzerland) at an excitation wavelength of 530 nm and an emission wavelength of 590 nm. Samples were tested in four replicates. Values of IC50, the concentration of sample required to inhibit cell growth by 50% in comparison with the growth of a cell control, were analysed using Origin 8.5.1 software (Origin Lab Corporation, USA) with a non-linear sigmoidal fit.
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