Cell proliferation was measured using the resazurin (AlamarBlue) fluorescence and Cell-Titer-Glo luminescence assays as previously described (6 (link),10 (link)). Drug synergy testing was performed by treating cells for 72 h with drug combinations of up to 1000 nM AZD6738 and 500 nM AZD1775. Survival was assayed by Cell-Titer-Glo and each experiment was repeated at least two times. The data were analyzed using SynergyFinder (https://synergyfinder.fimm.fi ) (19 (link)). For the dose response experiment, approximately 5 × 104 cells were plated per well of a 96-well plate in the presence or absence of doxycycline. Cells were treated with a range of AZD1775 concentrations for 72 hours. Fluorescence readings were obtained after adding the AlamarBlue reagent (Sigma) using a FLUOstar Omega microplate reader (BMG Labtech). IC50 values were then calculated using log-transformed and normalized data (GraphPad Prism 8.01).
Alamarblue reagent
Manufactured by Merck Group
Sourced in United States
AlamarBlue reagent is a colorimetric assay used to quantitatively measure cell proliferation, viability, and cytotoxicity. The reagent contains a non-toxic, cell-permeable compound that is reduced by metabolically active cells, changing the color of the reagent from blue to red. The degree of color change is proportional to the number of living cells.
Automatically generated - may contain errors
Lab products found in correlation
Fluostar omega microplate reader, by BMG Labtech (2 mentions)
Prism 8, by GraphPad (1 mentions)
Alamarblue reagent, by Thermo Fisher Scientific (1 mentions)
Glomax multi, by Promega (1 mentions)
Alamar blue assay, by Merck Group (1 mentions)
Fluostar omega, by BMG Labtech (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Bicinchoninic acid bca assay, by Merck Group (1 mentions)
Spectramax m5, by Molecular Devices (1 mentions)
Incucyte zoom, by Sartorius (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Faststart essential dna green master, by Roche (1 mentions)
Phalloidin conjugate solution, by Merck Group (1 mentions)
Anti rankl, by Cell Signaling Technology (1 mentions)
Anti opg, by Cell Signaling Technology (1 mentions)
Goat anti rabbit horseradish peroxidase conjugate, by Bio-Rad (1 mentions)
Trap solution, by Merck Group (1 mentions)
Anti tnf α, by Cell Signaling Technology (1 mentions)
Anti tgf β, by Cell Signaling Technology (1 mentions)
13 protocols using alamarblue reagent
1
Cell Proliferation and Drug Synergy Assays
2
Evaluating Leydig Cell Viability with AlamarBlue
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The viability of TM3 Leydig cells was evaluated using alamarBlue reagent (ThermoFisher Scientific, Invitrogen, Vantaa, Finland), as reported previously [27 (link),35 (link),36 (link)]. AlamarBlue assay is a sensitive oxidation-reduction indicator that fluoresces after the reduction of the blue color of resazurin to the pink color of resorufin, due to mitochondrial dehydrogenase activity in living cells [37 (link)]. In brief, Leydig cells were pre-cultured at an adjusted density (4 × 103 cells/well) in gelatin pre-coated 96-microwell plates, 24 h before the exposure. Afterward, the cell culture medium was removed, and fresh medium containing experimental doses (62.5–2000 µg/mL) of Lepidium sativum L. was applied for 24 h and 48 h. After the respective treatments, cells washed with Dulbeccos’s phosphate-buffered saline (DPBS; Sigma-Aldrich, St. Louis, MO, USA) were incubated with DMEM/F12 media (without phenol red) containing alamarBlue reagent in a final concentration of 5% (v/v). After 0.5 h min incubation in a CO2 incubator (37 °C; 5% CO2; and 95% atmospheric humidity), the fluorescence (excitation/emission: 530/590 nm wavelengths) was measured using a combined spectro-fluoro-luminometer GlomaxMulti+ (Promega Corporation, Madison, WI, USA).
3
Celosianin Cytotoxicity Evaluation via AlamarBlue
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Cell viability was assessed using the alamarBlue assay
(Sigma-Aldrich). H9c2 cells were seeded in 96-well plates at a density
of 5 × 1 × 103 cells/well for 24 h. The cells
were then treated with varying concentrations of celosianin and its
oxidized products 17-decarboxy-neocelosianin and 2,17-bidecarboxy-xanneocelosianin
(0.1 to 1000 μg/mL). Untreated cells served as the control (0
μg/mL pigment concentration). After 24 h of treatment, the culture
medium was removed and replaced with 100 μL of fresh DMEM containing
5% (v/v) alamarBlue reagent (Sigma-Aldrich). The
plates were incubated for 3 h, and then 100 μL of the supernatants
was transferred to a black 96-well plate. Fluorescence was measured
at an excitation wavelength of 544 nm and emission wavelength of 590
nm using a microplate reader (FluoStar Omega, BMG Labtech, Germany).
The percentage of resazurin reduction was calculated from the results
using the following formula where:
Fsample is the fluorescence value of the sample.
F0% red is the fluorescence of
a medium containing unreduced alamarBlue reagent.
F100% red is the fluorescence
of a medium containing reduced alamarBlue reagent.
(Sigma-Aldrich). H9c2 cells were seeded in 96-well plates at a density
of 5 × 1 × 103 cells/well for 24 h. The cells
were then treated with varying concentrations of celosianin and its
oxidized products 17-decarboxy-neocelosianin and 2,17-bidecarboxy-xanneocelosianin
(0.1 to 1000 μg/mL). Untreated cells served as the control (0
μg/mL pigment concentration). After 24 h of treatment, the culture
medium was removed and replaced with 100 μL of fresh DMEM containing
5% (v/v) alamarBlue reagent (Sigma-Aldrich). The
plates were incubated for 3 h, and then 100 μL of the supernatants
was transferred to a black 96-well plate. Fluorescence was measured
at an excitation wavelength of 544 nm and emission wavelength of 590
nm using a microplate reader (FluoStar Omega, BMG Labtech, Germany).
The percentage of resazurin reduction was calculated from the results
using the following formula where:
Fsample is the fluorescence value of the sample.
F0% red is the fluorescence of
a medium containing unreduced alamarBlue reagent.
F100% red is the fluorescence
of a medium containing reduced alamarBlue reagent.
4
Cell Viability Assay of Compounds
5
Evaluating Drug Sensitivity in Prostate Cancer
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Human and murine prostate cancer cell lines were obtained from the American Type Culture Collection (ATCC) with the exception of C42 cells which were a gift from Dr. Leland Chung. Generation of enzalutamide resistant LNCaP cells was achieved through maintenance in 10 μM enzalutamide in 5% FBS/DMEM for three months in parallel with control treated cells. Alamar Blue assays were performed by plating 4 × 10(3) cells per well over a 48 well plate. The following day cells were treated with various concentrations of OP449 (1–10 μM). Cells were exposed to drug for 3–4 days followed by the addition of alamar blue reagent (R70717, Sigma) for 2 hours for colorimetric reading. Generation of IC50 values was achieved using Prism Software.
6
Synthetic Lipoxin A4 Modulation of Immune Cells
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Synthetic lipoxin A4 (LXA4) was purchased from Cayman Chemical. Dulbecco's modified eagle's medium (DMEM/F‐12), RPMI‐1640 medium, penicillin–streptomycin (PS), and trypsin–EDTA solution were all purchased from Gibco®, Thermo Fisher Scientific. Fetal bovine serum (FBS), phosphate‐buffered saline (PBS) tablets, bovine serum albumin (BSA), alamarBlue™ reagent, Pierce™ IP lysis buffer, and bicinchoninic acid (BCA) assay were all purchased from Sigma‐Aldrich. Commercially available preparations of LPS from E. Coli were purchased from InvivoGen. TNFα and IL‐4 ELISA kits were purchased from R&D systems. Millicell® EZ 8‐well glass slides were purchased from Merk. All cell culture flasks and plates were purchased from Greiner Bio‐one.
7
Evaluating hBMSC Proliferation on Glass and TiO2
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To evaluate proliferation of hBMSCs at seven and 14 days on the two different surfaces (Glass and TiO2), we used Alamar Blue reagent (Sigma Aldrich). According to manufacturer instructions 1:10 dilution in serum free Dulbecco’s modified Eagle’s medium (DMEM) low glucose was added to cells and left for 3 h at 37 °C in 5% CO2. Then, absorbance of 100 μL at 595 nm was read and converted to cell numbers with a conversion curve. The cell viability was also performed at the end of culture conditions (28 days in PM and OM culture conditions).
8
Cell Viability Assessment with AlamarBlue
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The cell viability was assessed with AlamarBlue reagent (Sigma-Aldrich, St. Louis, MO). Cells were incubated with AlamarBlue solution at 10% for 3 h and measured fluorescence signal (excitation at 560 nm and emission at 590 nm) with SpectraMax-M5 (Molecular Devices, Sunnyvale, CA). OTX-015 (DC7150), IBET151 (DC5183), LY294002 (DC1058) and MK-2206 (DC7465) were all purchased from DC Chemicals (Shanghai, China). The cell images were collected by the IncuCyte ZOOM (Essen BioScience, Ann Arbor, MI).
9
Osteoclast Differentiation and Activation Assay
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α‐Minimal essential medium (α‐MEM), penicillin, streptomycin, foetal bovine serum (FBS), qPCR SuperMix UDG kit were purchased from Invitrogen (Carlsbad, CA). Lipopolysaccharides (LPS), macrophage monocyte colony‐stimulating factor (M‐CSF), methylthiazolyldiphenyl‐tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), TRAP solution, Acid Phosphatase Liquicolor Assay kit, Hoechst 33342, ELISA kits, alamar blue reagent and phalloidin conjugate solution were purchased from Sigma‐Aldrich. Anti‐NFATc1 (H‐110), anti‐Ap1 (Sc‐57761) and anti–c‐Fos (H‐125) monoclonal antibodies were purchased from Santa Cruz Biotechnology. Anti–TNF‐α, anti‐RANKL, anti‐OPG, anti–IL‐1β, anti–IL‐6 and anti–TGF‐β were purchased from Cell Signaling Technology. NucleoSpin for RNA extraction kit was purchased from Macherey–Nagel. Toluidine blue stain was purchased from BioGenex. FastStart Essential DNA Green Master was purchased from Roche. Goat anti‐rabbit horseradish peroxidase conjugate was purchased from Bio‐Rad.
10
Cell Proliferation Assay and Synergy Analysis
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Cell proliferation was measured using the resazurin (AlamarBlue) fluorescence assay [102 ]. Approximately 5 × 104 cells were plated per well of a 96-well plate. Cells were treated with a range of drug concentrations for 6-72 hours. Fluorescence readings were obtained after adding the AlamarBlue reagent (Sigma) using a FLUOstar Omega microplate reader (BMG Labtech). IC50 values were then calculated using log-transformed and normalized data (GraphPad Prism 5.0). The combination index (CI) as a measure of drug synergy was determined using the method of Chou and Talalay with six drug concentrations at a fixed dose ratio [72 (link)]. The data were analyzed using the CompuSyn software (http://www.combosyn.com/ ).
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