Clear bottom 96-well plates were seeded with 5×103 MCF-7 cells per well and treated with 0, 1, 10, or 100 ng/mL purified recombinant cmvIL-10 (R&D Systems) in triplicate. Cell supernatants were harvested at various time points for subsequent analysis. Cell lysates were also collected from these plates by adding 100μL cell lysis buffer (150mM NaCl, 20 mM HEPES, 0.5% Triton X-100, 1mM NaOV4, 1mM EDTA, 0.1% NaN3) with fresh protease inhibitor (Calbiochem, La Jolla, CA) onto the cells and administering a freeze-thaw cycle. MMP-10 protein levels were determined by sandwich ELISA according to manufacturer’s instructions (R&D Systems Duo Set DY910). Protein concentrations were determined by interpolation from a standard curve with an R2 value greater than 0.99 using the Opsys MR plate reader and Revelation Quicklink 4.24 software (Dynex Technologies, Chantilly, VA).
Opsys mr plate reader
Manufactured by Dynex
Sourced in United States
The Opsys MR plate reader is a multi-mode microplate reader designed for a variety of applications in life science research. It can perform absorbance, fluorescence, and luminescence measurements on microplates. The device is equipped with a monochromator-based optical system that enables flexible wavelength selection.
Automatically generated - may contain errors
Lab products found in correlation
Prism 6, by GraphPad (2 mentions)
Purified recombinant cmvil 10, by R&D Systems (1 mentions)
Quantikine elisa kit, by R&D Systems (1 mentions)
Dmem 10 fcs, by Thermo Fisher Scientific (1 mentions)
Dy999, by R&D Systems (1 mentions)
Baf293, by R&D Systems (1 mentions)
Microtiter plate, by Thermo Fisher Scientific (1 mentions)
A9544, by Merck Group (1 mentions)
9 protocols using opsys mr plate reader
1
Quantification of MMP-10 Secretion in MCF-7 Cells
2
Quantifying IL6 Levels in MLEC
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Mouse IL6 cytokine concentrations in MLEC treated with recombinant mIL6 and cediranib were measured using Quantikine ELISA kit (R&D Systems) according to the manufacturer's protocol. Absorbance was measured at 450 nm using an Opsys MR plate reader (Dynex Technologies).
3
Evaluating MSC Proliferation with Patients' Serum
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The effect of patient’s serum on MSC proliferation in vitro was assessed in a colorimetric cell proliferation assay based on a tetrazolium salt XTT (Roche, Welwyn Garden City, UK), as described previously [27 (link)]. In brief, the assay was performed using cultured MSCs from four donors, in quadruplicate for each cell seeding density and each serum sample. Cells were seeded at 125, 250 and 500 MSCs/well in 96-well plates and were allowed to attach for 24 hours in (D)MEM/2% FCS (both from Invitrogen, Paisley UK); the next day media were replaced with 150 μl of either non-haematopoietic (NH) media (positive control wells), (D)MEM/10% FCS not optimized for MSC growth (negative control wells) or (D)MEM/10% patient’s serum (test wells). MSCs were allowed to grow and the assay was stopped on day 5 by replacing the growth media with the XTT labelling mixture; the colour change was read at 450 and 620 nm using Opsys MR Plate reader (Dynex Technologies, Worthing, UK). Optical densities (ODs) were analysed separately for each seeding density and MSC culture and normalised to the OD of the positive control (NH media); the values for four MSC cultures were next averaged for each seeding density and serum proliferative indices (PIs <1 = less proliferative than NH, >1 = more proliferative than NH) were recorded for each time-point, for each serum.
4
MTT Assay for Cell Viability
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Cell viability was assayed by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) test according to [30 (link)]. After cell exposure to products, 96-well plates were removed from the incubator and to each well 20 μl of MTT solution was added in dim/dark conditions using a covered test tube (for the reagents). The plate was wrapped in aluminum foil and incubated for 2 h at 37 °C. Afterwards, the contents of each well was removed and 200 μl of lysis buffer was added to the wells, including blank wells that served for the calibration of the plate reader. The plate was read at 590 nm using an Opsys MR plate reader (Dynex Technologies inc., Chantilly, VA, USA). For treated cells, viability was expressed as a percentage in respect to the negative controls. Each treatment and each concentration was done in six replicates.
5
VEGF-A Quantification by Sandwich ELISA
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Immunoassay 96-well plates were coated with Exon8apab antibody (25 µg/ml in 1× PBS, 100 μl/well) and left overnight at room temperature. After washing in triplicate (0.05 % Tween® in 1× PBS, 200 µl/well), immunoassay plates were blocked (1 % Bovine Serum Albumin in 1× PBS, 200 µl/well) and incubated at 37 °C for minimum of 2 h. The plates were washed and samples added, using recombinant human VEGF-A165 (840164, R&D Systems) as a serial dilution control standard. Samples were assessed in triplicate (100 µl/well, diluted in 1 %BSA/PBS). The plate was then incubated at 37 °C for 2 h with shaking.
Following washing, biotinylated goat anti-human panVEGF-A detection antibody (BAF293, R&D Systems) was added at 100 ng/ml in 1 %BSA/PBS (100 µl/well). The plates were then incubated once more at 37 °C for 2 h. Following washing, HRP-conjugated streptavidin was added (1:200 in 1 %BSA/PBS, 100 µl/well, 890803, R&D Systems) and incubated without light exposure or agitation for 30 min at RT. The plates were washed once more, and HRP ELISA substrate (100 µl/well, DY999, R&D Systems) was added before a final incubation without light exposure at RT for 15–30 min. A stop solution (1 M H2SO4, 50 µl/well) was then added directly to the substrate and resultant colour change measured at 450 nm using an Opsys MR plate reader (Dynex, USA).
Following washing, biotinylated goat anti-human panVEGF-A detection antibody (BAF293, R&D Systems) was added at 100 ng/ml in 1 %BSA/PBS (100 µl/well). The plates were then incubated once more at 37 °C for 2 h. Following washing, HRP-conjugated streptavidin was added (1:200 in 1 %BSA/PBS, 100 µl/well, 890803, R&D Systems) and incubated without light exposure or agitation for 30 min at RT. The plates were washed once more, and HRP ELISA substrate (100 µl/well, DY999, R&D Systems) was added before a final incubation without light exposure at RT for 15–30 min. A stop solution (1 M H2SO4, 50 µl/well) was then added directly to the substrate and resultant colour change measured at 450 nm using an Opsys MR plate reader (Dynex, USA).
6
IgG Immune Response Quantification by ELISA
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The immunoglobulin G (IgG) immune response against the synthetic P27A and overlapping 20-mers was measured using an enzyme-linked immunosorbent assay (ELISA) (5 (link), 15 (link)). Microtiter plates (Nunc™ Maxisorp, Denmark) were coated at a concentration of 2 and 5 μg/mL, respectively, at RT for 1 h then blocked with PBS-3% milk for 1 h. Samples and controls were serially diluted in PBS-1.5% milk-0-05% Tween and added at 50 μL per well. Controls were present on each ELISA plate. Anti-hu IgG secondary antibodies conjugated to alkaline phosphatase (A-9544, Sigma, St. Louis, MO), diluted 1:1000 in PBS-1.5% milk-0-05% Tween, were added to each well. p-Nitrophenyl phosphate substrate tablets (Sigmafast™, Sigma, St. Louis, MO) were dissolved and added to each well. The optical density (OD) of each well was measured at 405 nm using an OpsysMR plate reader (Dynex Technologies). Results were expressed as AU/mL based on the standard curve obtained from the response of the positive control pool of sera to P27A.
7
Quantifying Serum SFRP4 in Systemic Sclerosis
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The concentration of SFRP4 present in the sera of SSc patients was determined using a commercially available sandwich immunoassay (SFRP4 ELISA, USCNK life science Inc., Wuhan, China) as per manufacturer’s instruction. In summary, a standard curve with a detection range between 1000 pg/mL and 15.6 pg/mL was prepared using the supplied recombinant protein and standard diluent. Sera samples and protein standards were added to the pre-coated 96 well ELISA plate and incubated for 2 h at 37 °C. Detection reagent A was added for 1 h at 37 °C and the plate washed 3 times using the supplied wash buffer. Detection reagent B was added for 30 min at 37 °C and followed by wash buffer 3 times. Substrate solution was added and catalysis proceeded for a period of up to 5 min at RT. The reaction was stopped using stop solution and the absorbance of each well was read at 450 nm using an OpsysMR plate reader (Dynex Technologies, Chantilly, VA, USA).
8
EZH2 Inhibitor Cytotoxicity Screening
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THP-1, hTERT and primary cells were cultured in 1 x 10 4 cells in 100 µl of Opti-MEM per well in 96 well plates. Cells were treated with each EZH2 inhibitor (EZH2i) to a final concentration ranging from 1 nM to 100 µM. Cultured cells were incubated in the presence or absence of the tested drug for 96 hours. Total cell viability (% cytotoxicity) was measured using Alamar blue assay as described previously [33] . Briefly, cells were incubated with 10% Alamar blue for 4 hours, then the absorbance at 570-620 nm was measured (Opsys MR Plate Reader, Dynex Technologies, Chantilly, Virginia). Cell viability (%) for THP-1 cells and control (hTERT) cells were calculated by normalizing the absorbance ratio of the treated cells to the vehicle control (DMSO). IC50 was calculated by GraphPad Prism 6.0 program (GraphPad Software, Inc; CA, USA).
9
Cytotoxicity Evaluation of EZH2 Inhibitors
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THP-1, hTERT and primary cells were cultured in 1 × 10 4 cells in 100 µl of Opti-MEM per well in 96 well plates. Cells were treated with each EZH2 inhibitor (EZH2i) to a nal concentration ranging from 1 nM to 100 µM. Cultured cells were incubated in the presence or absence of the tested drug for 96 hours. Total cell viability (% cytotoxicity) was measured using Alamar blue assay as described previously 33 . Brie y, cells were incubated with 10% Alamar blue for 4 hours, then the absorbance at 570-620 nm was measured (Opsys MR Plate Reader, Dynex Technologies, Chantilly, Virginia). Cell viability (%) for THP-1 cells and control (hTERT) cells were calculated by normalizing the absorbance ratio of the treated cells to the vehicle control (DMSO). IC50 was calculated by GraphPad Prism 6.0 program (GraphPad Software, Inc; CA, USA).
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