LN215 cells were cultured in 96-well plates to reach 90% confluence. The Fluo4 NW Calcium Assay Kit (F36206, Thermo Fisher Scientific) was used to monitor [Ca2+]in according to the manufacturer’s instructions. Growth media were removed and cells were incubated with 100 μL of the dye loading solution for 45 min at 37°C. Next, the cells were treated with vehicle or 50 μM FNZ and Fluo4 fluorescence was measured prior to and every 30 min after treatment with vehicle or FNZ for 240 min using a POLARstar microplate reader (BMG Labtech). As the Fluo4 dye loaded in the cells is released into culture medium at a millimolar Ca2+ concentration, Fluo4 fluorescence distinctly increases following 4 h of incubation. Thus, the Fluo4 fluorescence of FNZ-treated cells was normalized to that of vehicle-treated cells for each time point. To assess the rapid effect of FNZ on [Ca2+]in, the 96-well plate with cells was placed into a POLARstar microplate reader (BMG Labtech) after dye loading, as described above. Vehicle, 50 μM FNZ, or vehicle together with 100 μM ATP were added to each well using the automatic injector of the microplate reader and the Fluo4 fluorescence of each well was measured every 0.5 s for 100 s. The Fluo4 fluorescence of each group measured after the injection was normalized to that of the corresponding group at the time of injection initiation (0 s) and plotted as graphs.
Polarstar microplate reader
Manufactured by BMG Labtech
Sourced in Germany, Australia, United States
The POLARstar microplate reader is a versatile and highly sensitive instrument designed for a wide range of applications in life science research. It is capable of performing absorbance, fluorescence, and luminescence measurements on microplates, allowing researchers to conduct various assays and analyze samples efficiently.
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Lab products found in correlation
Amplex red assay kit, by Thermo Fisher Scientific (4 mentions)
Alamarblue cell viability assay, by Thermo Fisher Scientific (3 mentions)
Mtt assay, by Merck Group (2 mentions)
Lance ultra camp kit, by PerkinElmer (2 mentions)
Glutamine, by Merck Group (1 mentions)
Hepes, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Alamar blue solution, by Thermo Fisher Scientific (1 mentions)
Alamar blue, by Thermo Fisher Scientific (1 mentions)
Tg gpo pap kit, by Roche (1 mentions)
Horseradish peroxidase conjugated goat anti mouse igg, by Santa Cruz Biotechnology (1 mentions)
Tmb substrate, by Merck Group (1 mentions)
Fluo 4 nw calcium assay kit, by Thermo Fisher Scientific (1 mentions)
Mtt formazan powder, by Merck Group (1 mentions)
Prism 6, by GraphPad (1 mentions)
Adenosine deaminase ada, by Roche (1 mentions)
Accutase, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Forskolin, by Merck Group (1 mentions)
23 protocols using polarstar microplate reader
1
Monitoring Intracellular Calcium Dynamics
2
Toxin Neutralization Assay for TcdA and TcdB
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Toxin neutralization assays were performed as described previously (Roberts et al., 2012 (link)), with modification. Vero cells were seeded at 9 × 103 cells per well in 96-well black microtiter-plates (flat bottom, VWR) in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with Penicillin-Streptomycin (100 U/ml and 0.1 mg/ml, respectively; Sigma), Glutamine (2.0 mM; Sigma), HEPES (25 mM Sigma) and 10% Fetal Bovine Serum (Sigma), and incubated for 24 h at 37°C in a humidified incubator under 5% CO2. Purified TcdA or TcdB proteins were diluted in DMEM and prepared as 40 ng/ml or 400 pg/ml dilutions, respectively. Suitably diluted oral formulation, anti-TxA4 and anti-TxB4 antibodies were pre-mixed with either TcdA or TcdB in a 96-well culture plate and 100 μl of the pre-mix was transferred to the corresponding wells of the prepared plates with Vero cells. Plates were incubated at 37°C in 5% CO2 for 48 h. Each dilution was performed in duplicate. Forty μl of Cell-Titer blue stain (Promega) was added to each well and incubated for a further 4 h to determine cell viability. Fluorescence was determined using a POLARstar microplate reader (BMG Labtech) at λ590 nm. The cell survival percentage was calculated in Microsoft Excel and plotted for each concentration using GraphPad Prism. EC50 values were calculated using non-linear regression (Sigmoidal fit, 4PL).
3
Aromatase Protein Quantification in Tumors
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Enzyme-linked immunosorbent assay kits for human aromatase (ARO) (USCN Life Science Inc.) were used for aromatase protein detection. Protein was extracted from tumor and non-tumor samples, following this kit protocol. For this procedure, tissues were available from 85 patients. Protein concentration was measured in a Polar Star Microplate Reader (BMG Labtech) using the bicinchoninic acid protein method. Data were analyzed using Four Parameter Logistic Fit, developed by MyAssays, Analysis Software Solutions.
4
Cytotoxicity Evaluation of SLNs and CDDP Formulations
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DMEM medium was used to grow HeLa cell which is supplemented with 10% of FBS and 1% of antibiotic mixture in an incubator at 37 °C. The cytotoxicity assay was performed by MTT protocol. Briefly, 15,000 cells/well was seeded at each well of 96-well plate and let it aside for 24 h. The old media was removed and wells containing adherent cells were washed with PBS. The cells were incubated with SLNs (0.01–100 μg/mL) for 24 h. Also, free CDDP and CDDP-loaded formulations were exposed to cancer cells and incubated for 24 h. Following 24-h incubation, the old media containing free SLN and drug-loaded formulations were carefully aspirated and the cells were washed twice with PBS. This process was carried out in order to minimize the chance of carrier or drug that is not internalized by the cells and avoid any interference of the materials on the final absorbance. The cells were then added with 10 μl of MTT (5 mg/ml) and incubated for 4 h. Then MTT solution was removed or aspirated carefully, and cells were added with 150 μl of DMSO to solubilize the formazan crystals. The well plates were then kept aside for 15 min, and absorbance was studied at 570 nm using a POLAR star microplate reader (Omega, BMG LabTech). The IC50 value was calculated from using GraphPad prism software.
5
Gap Junction Communication Assay for Cell Lines
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The I-YFP GJIC assay was performed as previously described [17 (link)], with minor modifications. Briefly, a 1:4 mixture of LN215-YFP and LN215-SLC26A4 cells was plated on a 96-well plate at a density of 20,000 cells/well and incubated for 24 h. Culture media were aspirated and cells were washed twice with 200 μL of C-solution. Next, the cells were treated with vehicle or chemicals diluted in 100 μL C-solution and further incubated for the indicated period. The 96-well plate containing the cells was placed into a POLARstar microplate reader (BMG Labtech, Ortenberg, Germany). An equal volume of I-solution (10 mM HEPES [pH 7.4], 140 mM NaI, 10 mM glucose, 5 mM KCl, 1 mM MgCl2, and 1 mM CaCl2) was injected into each well at 1 s after each measurement was started at a rate of 135 μL/s using the machine-equipped automated injector. Fluorescence was measured for 20 s at 0.4 s intervals in kinetic mode using a 485 nm excitation/520 nm emission filter. The percentage (%) of YFP quenching and GJIC activity were calculated as follows:
6
Growth of GFP-Expressing Human ASCs in 3D Collagen
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The growth of GFP-expressed human ASCs cultured in 3D collagen scaffolds or on collagen coated (0.5 mg/mL; Invitrogen) plate was assessed by using alamarBlue assay. Briefly, GFP-expressed human ASCs (1x106 cells) were seeded in collagen scaffolds as 3D culture or in 12-well tissue culture plates as 2D culture, and cultured over a period of 3 weeks. At day 0, 2, 7, 14 and 21, scaffolds were cut into small pieces and incubated with alamarBlue® solution (Invitrogen) in a proportion of 1:10 (10 μL of alamarBlue® reagent to 100 μL of culture media) for 1 hour at 37°C in a humidified atmosphere containing 5% CO2. Fluorescence signals were measured at an excitation wavelength at 530–560 nm and an emission wavelength at 590 nm using a polarstar microplate reader (BMG Labtech, Australia) at 37°C.
7
Enzymatic Assay for β-Hexosaminidase Release
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For detection of the granular enzyme β-hexosaminidase, an enzymatic colorimetric assay was used as previously described.18 (link) Briefly, after activation of 100 µL of MCs in HEPES degranulation buffer, 50 µL of supernatant was transferred to a 96-well plate and mixed with 100 µL of substrate solution (3.5 mg/mL p-nitrophenyl-N-acetyl-β-D-glucosaminide dissolved in 40 mM citric acid, pH 4.5). The remaining cells (50 µL) were lysed with 150 µL of 0.1% triton X-100 and the same procedure was done. The mixtures were incubated at 37℃ for 90 minutes. After incubation, 100 µL of glycine (400 mM, pH 10.7) was added to each well, and the absorbance at 405 nm was measured using a POLAR-star Microplate Reader (BMG LabTech, Ortenberg, Germany). The percentage of β-hexosaminidase release was calculated as a percentage of the total β-hexosaminidase content as follows:
8
Liver Triglyceride Extraction and Quantification
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Liver triglycerides (TG) were extracted by the method of Folch and determined by a TG GPO-PAP kit (Roche Diagnostic, Australia) as previously described [18 (link)]. Briefly, 30–40mg of each liver sample was homogenized in 4 ml of chloroform/methanol (2:1) using a glass pestle tissue grinder. After the homogenization, the samples were rotated at room temperature overnight to ensure the complete solubilisation of the liver TG. The next day, 2 ml of 0.6% NaCl was added to each sample and followed by centrifugation to separate the aqueous from the organic phases. The lower chloroform layer contained liver TG were carefully transferred into a glass vial and dried completely under the nitrogen or air at 45°C. The extract was reconstituted in absolute ethanol for the determination TG using a POLARstar microplate reader (BMG Labtech, Germany).
9
Quantifying Gap Junction Intercellular Communication
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Donor and receptor cells were detached with trypsin, counted, mixed, and plated on 96-well plates. After 24 h of incubation, culture media were changed to 100 μL of C-solution (10 mM HEPES, pH 7.4, 140 mM NaCl, 10 mM glucose, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2). Vehicle or 25 μM CBX was used to treat cultures for 10 min when drug treatment was indicated. The GJIC assay was performed well by well. The fluorescence of each well was measured with a POLARstar microplate reader (BMG Labtech, Germany) every 0.4 s for the indicated time. One second after the first measurement, 100 μL of I-solution (10 mM HEPES, pH 7.4, 140 mM NaI, 10 mM glucose, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2) was added via the injector in the plate reader. The percent YFPQL fluorescence to time zero was calculated. The YFPQL quenching rate at a selected time point reflected GJ activity.
10
Influenza A Antibody Titration Assay
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Anti-haemagglutinin (HA) antibody titres in the sera were determined using the R&D indirect ELISA system (R&D Systems, Minneapolis, MN) with modification. Briefly, 96 well U-bottom vinyl non-treated Serocluster microplates (Costar, Corning NY) were coated with 100 ng Influenza A H1N1 (A/PR/8/1934) HA1 protein (Sino Biological, PR China) per well overnight at room temperature. All subsequent incubations were carried out at room temperature and plates were washed thrice with complete removal of residual fluid between incubation steps. Two-fold serially diluted sera were added in duplicates and incubated for 2 hours. After washing, horseradish peroxidase-conjugated goat anti-mouse IgG (Santa Cruz, CA) was added at 1:12000 for 2 hours. TMB substrate (Sigma-Aldrich Australia) was added and optical density read off a PolarStar microplate reader (BMG Labtech, Germany). The antibody titre was determined as the reciprocal of the highest dilution of the sample that yielded a reading of 2 SD above the mean of uninfected sera. To determine the level of anti-influenza neutralising antibodies, heat-inactivated and adsorbed sera from uninfected control and infected mice were assayed for haemagglutination inhibition (HI) as described [57 ].
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