Triglycerides (TG) from cell lines as well as tumor tissues were measured using Triglyceride-Glo Assay (Promega). In this assay, TG levels are determined by measuring Glycerol that is released from Triglycerides in an enzymatic reaction with a lipase. One molecule of glycerol per molecule if TG is released by this reaction. Glycerol is then measured in a coupled reaction that links the production of NADH to activation of proluciferin that produces light by luciferase activity. The amount of TG is determined from difference in glycerol measured in absence (free glycerol) and presence (total glycerol) of lipase. TG assay was performed according to manufacturer’s protocols. MCF7, T47D and MDA-MB-231 cells were seeded in 96 well plates, subsequently treated with different pH media with or without inhibitors. 48 h after treatments, cell was lysed with or without lipase and released glycerol was measured using detection reagent. Luminescence was read using the Flexstation3 Multimode Plate Reader (Molecular Devices). For measuring TGs from tumor tissues, 10 mg of cryopreserved tumor tissue was lysed in 250 μL of glycerol lysis buffer and diluted with lysis butter with or without lipase. TG levels were determined as the difference between total (with lipase) and free glycerol (without lipase).
Flexstation3 multimode plate reader
Manufactured by Molecular Devices
Sourced in United States
The Flexstation3 Multimode Plate Reader is a versatile laboratory instrument designed for high-throughput screening and assay development. It offers a range of detection modes, including fluorescence, luminescence, and absorbance, enabling users to perform a variety of assays within a single platform.
Automatically generated - may contain errors
Lab products found in correlation
Triglyceride glo assay, by Promega (2 mentions)
Phospho stat5 tyr694 homogeneous time resolved fluorescence cellular kit, by PerkinElmer (2 mentions)
Prism software 6, by GraphPad (1 mentions)
Fluo 4 am, by Thermo Fisher Scientific (1 mentions)
Daf fm diacetate, by Thermo Fisher Scientific (1 mentions)
Prestoblue reagent, by Thermo Fisher Scientific (1 mentions)
Flipr calcium 6 dye, by Molecular Devices (1 mentions)
12 protocols using flexstation3 multimode plate reader
1
Quantifying Cellular and Tumor Triglycerides
2
Quantifying STAT5 Phosphorylation by HTRF
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The phosphorylation of STAT5 was measured by the Phospho-STAT5 (Tyr694) Homogeneous Time Resolved Fluorescence (HTRF) cellular kit (Cisbio) according to the manufacturer protocol using the Flexstation 3 Multimode plate reader (Molecular Devices).
3
Fluorescein-Peptide Binding Kinetics to Akt2
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A total of 50 nM fluorescein-conjugated peptide was incubated with serial dilutions of Akt2 protein in the binding buffer at room temperature. Binding activity at 40 min was measured by fluorescence anisotropy using Flexstation 3 Multi-mode Plate Reader (Molecular Devices). KD values for FITC-labeled peptide binding to pAkt2 were determined by nonlinear regression analysis using the standard anisotropy equation in Prism software 6.0 (GraphPad).
4
Quantifying Cellular and Tumor Triglycerides
5
Quantification of UDP in Synovial Fluids
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Peripheral blood samples from healthy donors (No. H1 to No. H36, n = 36) and RA patients (Patient No. R1 to No. R36, n = 36) were collected into pyrogen-free and endotoxin-free test tubes with anticoagulants. Synovial fluids from OA (Patient No. O1 to No. O36, n = 36) or RA patients (Patient No. R1 to No. R36, n = 36) were added to an equal volume of PBS. Rat synovial fluids and peripheral blood were collected as described above. These samples were centrifuged at 1,000 ×g for 20 min at 4°C, and the supernatant was carefully collected. The UDP content in the samples was measured using a Transcreener UDP Assay (BellBrook Labs, United States) via a fluorescence polarization readout according to the manufacturer’s protocol. A 15 μL mixture of reagents, including 8 nm UDP2 antibody-Tb, 1× Stop & Detect Buffer C and UDP HiLyte647 Tracer, was mixed with 5 μL of each sample in a 96-well plate. The plate was incubated for 1.5 h at room temperature and analyzed in a FlexStation® 3 Multimode Plate Reader (Molecular Devices, United States). The concentration of UDP was calculated by the standard curve prepared with standard UDP solution before analysis.
6
Optical Intensity Measurement with Flexstation 3
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A spectrometer (Flexstation 3 multimode plate reader, Molecular Devices, Sunnyvale, CA, USA) was used to measure the optical intensity.
7
Quantifying mast cell degranulation
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LAD2 cells were washed twice and resuspended in 0.1% SIR-BSA. Fourty-five microliters of cells (0.45 × 106 cells/mL) were seeded and stimulated with CST-14, LL-37, substance P, compound 48–80, or (R)-Zinc-3573 for 25 min. For total β-hexosaminidase release, cells were lysed using 0.1% Triton X-100. The supernatant (20 μL) was collected and incubated with an equivalent volume of 4 mM p-nitrophenyl-N-acetyl-β-D-glucosamine (PNAG) for 1 h at 37°C. The reactions were halted through the addition of 0.1 M NaHCO3/0.1 M Na2CO3 buffer. The β-hexosaminidase release assay for human skin mast cells was performed as described previously by McHale et al. (39 (link)). For assays using inhibitors, cells were incubated for 30 min with the appropriate drug prior to agonist stimulations. Inhibitor concentrations were determined using IC50 values reported by the drug manufacturer. Absorbance was measured using FlexStation® 3 multi-mode plate reader (Molecular Devices; San Jose, CA) at 405 nm. Percent of β-hexosaminidase release content was calculated by dividing absorbances of agonist-stimulated cells by total cell β-hexosaminidase content.
8
High-throughput calcium flux assay in cells
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A collagen layer was coated rotating overnight at 4 °C on a clear bottom 96-well black plate (Corning 3603) as previously described [21 (link)]. Cells were then plated in the prepared plate at 50,000 cells per well for a confluent monolayer. Cells were then loaded with 4 μM Fluo4-AM (Life Technologies F14201) and prepared as previously described [21 (link)] prior to the addition of compound. The final volume per well was 100 μL of Hank’s Balanced Salt Solution (HBSS + Ca2+; Gibco 14025-092).
Dilutions of compounds were prepared in a separate plate at 5× concentration for the robotic addition of 25 μL to cells by the FLEX Station 3 Multi-Mode plate reader (Molecular Devices). The FLEX Station 3 reads Relative Fluorescence Units (RFUs) measured every 1.28 s for a period of 300 s after compound addition. The results were either plotted as the maximum-minimum for each compound concentration over the time period or ΔF/F0 as previously calculated [21 (link)] with the initial 30 s reading as a baseline value prior to compound addition. All values shown are mean SD of triplicate samples.
Dilutions of compounds were prepared in a separate plate at 5× concentration for the robotic addition of 25 μL to cells by the FLEX Station 3 Multi-Mode plate reader (Molecular Devices). The FLEX Station 3 reads Relative Fluorescence Units (RFUs) measured every 1.28 s for a period of 300 s after compound addition. The results were either plotted as the maximum-minimum for each compound concentration over the time period or ΔF/F0 as previously calculated [21 (link)] with the initial 30 s reading as a baseline value prior to compound addition. All values shown are mean SD of triplicate samples.
9
Quantifying STAT5 Phosphorylation by HTRF
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The phosphorylation of STAT5 was measured by the Phospho-STAT5 (Tyr694) Homogeneous Time Resolved Fluorescence (HTRF) cellular kit (Cisbio) according to the manufacturer protocol using the Flexstation 3 Multimode plate reader (Molecular Devices).
10
Intracellular Nitric Oxide Quantification
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Intracellular NO was detected by using DAF-FM diacetate (Invitrogen, Carlsbad, CA, USA) as previously described63 (link). Briefly, hCSs and iCSs were treated in presence of DOX with or without L-NIO for 24 h in hanging drop plates. Spheroids were then transferred in clear bottom black 96 well plates by centrifugation. Spheroids were rinsed twice with PBS and 1 μM DAF-FM diacetate solution was prepared freshly in PBS. After 30 min, DAF-FM diacetate solution was removed, spheroids were rinsed twice with PBS and fresh PBS was added for 30 min. For measurements of NO synthesis, the microplate was placed in the FlexStation® 3 Multimode Plate Reader (Molecular Devices, Sunnyvale, CA, USA) and measurements were recorded at 515 nm. NO synthesis was calculated by normalizing measurements against total number of cells and then against control (media only without DOX and/or L-NIO), and analysed using GraphPad Prism™ (La Jolla, CA).
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