TC28a2 cells were cultured overnight at a density of 30,000 cells per well in a 24-well plate for image analysis or 10,000 cells per well in a 96-well plate for quantification using GloMax®-Multi+ Detection System (Promega, Madison, WI, USA). Cells were then serum starved overnight. Fluo8AM (Invitrogen) was diluted to 5 mM in DMSO. The Fluo 8AM was then diluted to 5 µM in DMEM before addition to cells. Cells were incubated in 5 µM Fluo8 for 30 min before washing with DMEM three times. Cells were incubated for a further 30 min prior to fluorescent imaging with the Leica DFC365 FX microscope with the Leica Application Suite (Leica Wetzlar, Germany) or fluorescence detection on the GloMax®-Multi+ Detection System (Promega).
Glomax multi detection system
Manufactured by Promega
Sourced in United States, Italy, United Kingdom, Germany, Japan, Spain, France
The GloMax-Multi Detection System is a multi-mode microplate reader designed for a variety of luminescence, fluorescence, and absorbance-based assays. It provides accurate and reliable detection of different types of signals from various sample types.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (60 mentions)
Dual luciferase reporter assay system, by Promega (58 mentions)
Dual glo luciferase assay system, by Promega (28 mentions)
Caspase glo 3 7 assay, by Promega (28 mentions)
Celltiter glo luminescent cell viability assay, by Promega (27 mentions)
Passive lysis buffer (plb), by Promega (23 mentions)
Celltiter glo, by Promega (16 mentions)
Opti mem, by Thermo Fisher Scientific (13 mentions)
Caspase glo 3 7 assay kit, by Promega (12 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (12 mentions)
Dual luciferase reporter assay, by Promega (11 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (10 mentions)
Luciferase assay system, by Promega (9 mentions)
Celltiter glo reagent, by Promega (9 mentions)
Celltiter glo luminescent cell viability assay kit, by Promega (9 mentions)
Fugene hd, by Promega (8 mentions)
Luciferase assay reagent, by Promega (8 mentions)
Nano glo dual luciferase reporter assay system, by Promega (8 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (8 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (8 mentions)
1 190 protocols using glomax multi detection system
1
Calcium Imaging of TC28a2 Cells
2
Biomass, Protein, and GFP-SncA Analysis
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From shake flask cultures, 4 mL of samples were taken at the indicated time point. Biomass and culture supernatant were separated by suction filtration under vacuum. Biomass was collected, frozen at − 80 °C, and freeze dried overnight to determine biomass yield (dry weight). Total protein secretion in the culture supernatant was determined via the Bradford assay (BioRad) according to the manufacturers’ protocols and absorbance (600 nm) was measured using a GloMax®-Multi Detection System (Promega). Quantification of residual glucose in the cultivation medium was performed with the Glucose GOP/PAP Liquicolor kit (Human, Germany) according to the manufacturer’s manual. Total GFP-SncA fluorescence was determined in freeze dried biomass. 50 mg dried biomass were grinded and resuspended in 1 mL 50 mM NaPO4 buffer pH 7.0. Following ultrasonification for 10 min, fluorescence signal in supernatant was determined using a GloMax®-Multi Detection System (Promega) equipped with a blue filter (excitation: 490 nm, emission: 510–570 nm).
3
Mitochondrial Toxicity Assay for Worms
4
Nematode ATP Production Assay
5
In Vitro and Cellular Translation Assays
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The translation of ORFs were detected using both in vitro translation assays (IVT assays) and cultured cells. For IVT assays, TnT® Quick Coupled Transcription/Translation System was used for in vitro translation of all HiBiT-tagged ORFs. According to the manufacturer's instructions, 1 μg of plasmid DNA was used as template for each reaction and the reaction was incubated for 90 min at 30°C. The detection of translated products was performed using Nano-Glo® HiBiT Lytic Detection System (Promega, #N3030) according to the manufacturer's instructions with minor modifications. In brief, 10 μl IVT products were diluted to 50 μl using nuclease free water, added with 50 μl lytic buffer and mixed by pipet, after 10 minutes incubation at room temperature, the luminescence was measured on a Promega GloMax®-Multi Detection System. To further explore the translational potential of the candidates in cellular context, 500 ng of plasmid DNA was transfected into N2A, Hela and ARPE19 cells using Lipofectamine 3000. Cells were harvested at 48 h post-transfection for subsequent analysis. The plate was equilibrated to room temperature, then 300 μl lytic buffer was added to each well and incubated for 10 min on orbital plate shaker. The lysates were divided into three tubes and the luminescence of each tube was measured on a Promega GloMax®-Multi Detection System.
6
Cytotoxicity and Viability Multiplexing
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Commercially available cytotoxicity (CellTox™ Green) and ATP content based (CellTiter-Glo®) assays (Promega GmbH, Walldorf, Germany) were multiplexed in a single 96 well-plate following the manufacturer’s instructions. For positive controls, lysis solution 25× was added in wells containing vehicle control treated cells (0.5% DMSO). Fluorescence was measured at λex = 485–500 nm/λem = 520–530 nm in the GloMax®-Multi Detection System (Promega). Luminescence was measured in the GloMax®-Multi Detection System (Promega) and was normalized to the values of the vehicle control. Cytotoxicity and ATP cell viability analysis were carried out for range finder pre-tests and in parallel with metabolomics experiments in plates handled and treated exactly as the ones used for metabolite profiling.
7
Piceatannol's Impact on Oxidative Stress
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To proceed with all the subsequent analyses of oxidative stress two different concentrations of piceatannol were chose based on cell viability and general metabolism results. hPLF were seeded at a density of 2 × 104 cells/well in 96-well culture plates and after 24 h, they were treated during 1 h with 0.1 and 1 µM of piceatannol in the presence or absence of H2O2 (200 μM). The membrane integrity and ATP production were analyzed through the mitochondrial tox-glo™ Assay (Promega, Madison, WI, USA) according to the manufacturer’s instruction. Cells were incubated with cytotoxicity reagent at 37 °C for 30 min and fluorescence was measured at 520 nm (Glomax® multi detection system— Promega Corporation, Madison, WI, USA). This value represents the membrane integrity (MI). Thereafter the same plate was equilibrated at room temperature and then incubated with the ATP detection substrate; luminescence was measured after 5 min to 1 h for the detection of ATP production (Glomax® multi detection system—Promega Corporation, Madison, WI, USA). All analysis was performed in triplicate and vehicle (methanol 20%) was also tested.
8
Measurement of Metabolic Activities
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Metabolic activities were measured using calcein acetoxymethyl ester. After incubation of cells with 1 μmol/L calcein acetoxymethyl ester for 30 minutes at 37°C, fluorescence was measured on the GloMax‐Multi+ Detection System (Promega). Concentrations of intracellular pyruvate or pyruvate in the medium were measured using a Pyruvate Colorimetric/Fluorometric assay kit (BioVision, Milpitas, CA, USA) in accordance with the protocol from the manufacturer. Absorbance was measured on the GloMax‐Multi+ Detection System (Promega). To inhibit MCT, α‐cyano‐4‐hydroxycinnamic acid (4‐CIN) (Sigma‐Aldrich) was used. Cells were incubated with 500 μmol/L 4‐CIN for 12 hours at 37°C, then cells were evaluated by appropriate procedures for each assay.
9
Characterizing Cell Proliferation and Viability
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To create growth curves, cells were seeded in 96-well plates (1 × 103 per well) in DMEM medium with and without glucose. Cells were harvested daily for six consecutive days using a luminometric assay performed according to the manufacturer's protocol (CellTiter-Glo Luminescent Cell Viability Assay, Promega, Madison, WI). Luminescence was measured after 15 min incubation with the luminescent substrate and scanned using Glomax Multi Detection System (Promega). Relative viability was calculated as described before [73 (link)]. Graphs were generated using GraphPad Prism version 6 software (San Diego, CA). Cell viability was also assessed by crystal violet staining. For low seeding density conditions, cells were seeded at the density 3 × 103 cells per 35 mm well of a six-well plate and cultured for 2–5 wk in DMEM medium with and without glucose. Cells were later washed with PBS, fixed in 4% formaldehyde and stained with 0.1% crystal violet. Quantitation was performed by extracting the crystal violet dye with 10% acetic acid and measuring the absorbance at 550 nm with the Glomax Multi Detection System (Promega). At least two independent experiments were performed in triplicate for each cell line for all proliferation and viability assays. All cells were harvested at ≤ 80% confluency.
10
Quantitative Cell Migration and Colony Formation Assays
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The cell migration assay was performed using polycarbonate membrane with 8 μm pore size (Cell Biolabs, cat. CAB-100). 2 × 105 cells in the serum free medium were added to the inside of each membrane, FBS-containing medium was added to each matched well on 24-well plate. After 48 hour incubation, noninvasive cells were removed from the upper surface of the transwell membrane, and cells which migrated through the membrane were incubated with Cell Stain Solution, washed with Extraction Solution, absorbance at 560 nm was measured using GloMax-Multi Detection System (Promega).
For colony formation assay 5 × 103 cells were seeded on 6-well soft agar plate, maintained at 37°C for 10–20 days, washed with PBS and stained with Giemsa solution. The number of colonies (containing >50 cells) was counted using the following formula: colony formation efficiency = number of colonies/number of cells seeded.
MTS cell proliferation assay was performed following manufacturer instructions (Promega Corp., cat G3581). Cells were plated in 96-well plates and absorbance values were obtained using a GloMax multi detection system (Promega).
For colony formation assay 5 × 103 cells were seeded on 6-well soft agar plate, maintained at 37°C for 10–20 days, washed with PBS and stained with Giemsa solution. The number of colonies (containing >50 cells) was counted using the following formula: colony formation efficiency = number of colonies/number of cells seeded.
MTS cell proliferation assay was performed following manufacturer instructions (Promega Corp., cat G3581). Cells were plated in 96-well plates and absorbance values were obtained using a GloMax multi detection system (Promega).
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