Cytotoxic activity was measured by the LDH Cytotoxicity Assay Kit (BioVision, Linda, CA, USA). Cells (5 × 10 3 /100 µL) in each well on 96-well plates were incubated and centrifuged at 250g for 10 min. Supernatant of 100 µL was transferred in clear 96-well plates. After addition of reaction mixture (2.5 µL catalyst solution in 112.5 µL dye solution), cells were incubated for 30 min at room temperature. Absorbance was measured using GloMax TM Discover System GM3000 (Promega). All experiments were performed in triplicate.
Glomax discover system gm3000
Manufactured by Promega
Sourced in United States
The GloMax Discover System GM3000 is a multimode microplate reader designed for a variety of assays and sample types. It offers luminescence, fluorescence, and absorbance detection capabilities.
Automatically generated - may contain errors
Lab products found in correlation
Ldh cytotoxicity assay kit, by Abcam (2 mentions)
Ros glo h2o2 assay kit, by Promega (2 mentions)
Multitox glo multiplex cytotoxicity assay kit, by Promega (2 mentions)
Pmirglo e1330, by Promega (1 mentions)
Celltiter glo luminescent cell viability assay, by Promega (1 mentions)
Mitochondria isolation kit, by Abcam (1 mentions)
Complex 1 enzyme activity assay kit, by Abcam (1 mentions)
Cck 8 assay kit, by Dojindo Laboratories (1 mentions)
14 protocols using glomax discover system gm3000
1
LDH Cytotoxicity Assay Protocol
2
ROS Production Measurement by H2O2 Assay
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ROS production was measured by the ROS-Glo H 2 O 2 Assay Kit (Promega). Cells (1 × 10 4 /mL) in each well on 96-well plates were incubated and treated with H 2 O 2 substrate solution (25 µM/well), and reincubated at 37°C. After addition of ROS-Glo Detection solution (100 µL/ well), cells were incubated for 20 min at room temperature. Absorbance was measured using GloMax TM Discover System GM3000 (Promega). All experiments were performed in triplicate.
3
Multiplexed Cytotoxicity Assay Protocol
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Cells (5 × 10 3 /100 µL) were seeded in 96-well plates, and reagents of the MultiTox-Glo Multiplex Cytotoxicity Assay Kit (Promega) were added to cells after treatments as indicated by manufacturer's protocol. 27 Fluorescent and luminescent values were measured using GloMax TM Discover System GM3000 (Promega). Viability was calculated as a ratio of live/dead cells and expressed as percentage of untreated cells. All experiments were performed in triplicate.
4
Quantifying Cellular ROS Levels
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ROS production was measured by the ROS-Glo H 2 O 2 Assay Kit (Promega). Cells (1 × 10 4 /mL) in each well on 96-well plates were incubated and treated with H 2 O 2 substrate solution (25 µM/well) and incubated at 37°C. After addition of ROS-Glo detection solution (100 µL/well), cells were incubated for 20 min at room temperature. Absorbance was measured using GloMax TM Discover System GM3000 (Promega). All experiments were performed in triplicate.
5
LDH Cytotoxicity Assay Protocol
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Cytotoxic activity was measured by the LDH Cytotoxicity Assay Kit (BioVision, Linda, CA, USA). Cells (5 × 10 3 /100 µL) in each well on 96-well plates were incubated and centrifuged at 250g for 10 min. Supernatant of 100 µL was transferred in clear 96-well plates. After addition of reaction mixture (2.5 µL of catalyst solution in 112.5 µL of dye solution), cells were incubated for 30 min at room temperature. Absorbance was measured using GloMax TM Discover System GM3000 (Promega). All experiments were performed in triplicate.
6
Multiplex Cytotoxicity Assay for Cell Viability
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Cells (5 × 10 3 /100 µL) were seeded in 96-well plates, and reagents of the MultiTox-Glo Multiplex Cytotoxicity Assay Kit (Promega) were added to cells after treatments as indicated by manufacturer's protocol. 26 Fluorescent and luminescent values were measured using GloMax TM Discover System GM3000 (Promega). Viability was calculated as a ratio of live/dead cells and expressed as percentage of untreated cells. All experiments were performed in triplicate.
7
Identifying miR-335-3p Target Gene COPB2 via Luciferase Assay
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The bioinformatics website TargetScan 7.2 (www.targetscan.org ) was used to identify COPB2 as the target gene of miR-335-3p. For the luciferase reporter assay, the cDNAs of wild-type COPB2-3−UTR (5−-TTTCCTTTCTCAATAATGAAAAT-3−) and COPB2-3−UTR-mut (5−-TTTCCTTTCTCAATATACTTTTT-3−) were synthesized by TsingKe Biotech Co., Ltd. (Beijing, China) and cloned into the luciferase reporter gene vector pmirGLO (E1330) (Promega, Madison, WI, USA) to construct the COPB2-3−-UTR plasmid and the COPB2-3−-UTR-mut plasmid. Firefly luciferase was used as the reporter for miRNA regulation of the 3−UTR. Briefly, the NCI-H1975 cells were maintained in 96-well plates and co-transfected with a luciferase reporter plasmid (0.05 μg/well) and mimic (5 pmol). Cells were divided into four groups: cells transfected with control mimic and COPB2-3−UTR plasmid; cells transfected with miR-335-3p mimic COPB2-3−UTR plasmid; cells transfected with miR-335-3p mimic and COPB2-3−UTR-mut plasmid; and cells transfected with control mimic and COPB2-3−UTR-mut plasmid. After incubation at 37°C for 72 h, the firefly luciferase activity was measured by using the luciferase reporter assay system (N1610) (Promega, Madison, WI, USA) with a GloMax Discover System (GM3000) (Promega, Madison, WI, USA). The relative firefly reporter activity was calculated through normalization to the Renilla luciferase control.
8
Quantifying Cellular ATP and Metabolites
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The CellTiter-Glo® Luminescent Cell Viability Assay (G9241 Promega, WI, USA) was used to quantify a standard curve of different concentration of an ATP solution. Add a volume of CellTiter-Glo® 2.0 Reagent equal to the volume of medium containing cells. The contents were then mixed for 2 min on an orbital shaker to induce cell lysis. The plate was incubated at room temperature for 10 min to stabilize the luminescent signal, and the luminescence was recorded. The levels of glucose, glutamate, and lactate were determined using a glucose, glutamate, or lactate Glo assay kit (J6021, J5021, and J7021, Promega). Both X-ray (-) and X-ray ( +) cells were washed in cold PBS, immediately inactivated with 0.6 N HCl to halt metabolic activity, and neutralized with 1 M Tris base. The resulting lysate was combined at a 1:1 ratio with each detection reagent and luminescence was measured using a microplate reader (GloMax® Discover System, GM3000, Promega).
9
Antimicrobial Activity of AgNP Formulations
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Antimicrobial activities were measured for E. coli with the cell number of 1.5 × 108 CFU mL−1. All samples were incubated in an orbital shaking incubator (37 °C and 180 rpm) for 18 h. LB broth medium and bacteria alone served as the blank and the negative control group, respectively. The experimental groups included bacteria incubated with 5 mg mL−1 BSA or LA in LB broth as well as bacteria incubated with 62.5–500 µg mL−1 non-oxidized AgNP, oxidized AgNP, and AgNP@LA in LB broth. Additionally, time-dependent activities were assessed for bacteria incubated with 250 µg mL−1 AgNP@LA and Ag-ND@BSA from 1 h to 36 days. The BacTiter-Glo microbial cell assay (Promega), which measured the cell activity through a mono-oxygenation of luciferin catalyzed by firefly luciferase (an ATP-dependent enzyme), was used to assess the bacterial ATP levels. To avoid any possible interference by the nanoparticles, AgNP, AgNP@LA, and Ag-ND@BSA were removed by LB wash through centrifugal separation prior to the assays. A multimode luminometer (GloMax Discover System GM3000, Promega) measured the luminescence produced from the luciferase-catalyzed oxidative reactions.
10
Mitochondrial Complex I Activity Assay
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The left ventricle specimens for mitochondrial complex I enzyme activity analysis were immediately frozen to − 80 °C. Mitochondria were extracted from the specimens using a Mitochondria Isolation Kit (ab 110168, Abcam). The protein concentrations of the extracts were determined by Bicinchoninic Acid protein assay and adjusted to 100 µg/mL with PBS. Subsequently, a Complex I Enzyme Activity Assay Kit (ab109721, Abcam) was used to determine the activity of the electron transport chain (ETC) following the manufacturer’s protocol. The absorbance at 450 nm was measured using a microplate reader (GloMax Discover System GM3000, Promega Corporation, USA).
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