Cardiomyocytes were cultured on either 15 kPa PAA gels or glass coverslips at a density of 1.5 × 105 cells per sample in DMEM medium supplemented with 5% FBS, 10 mM HEPES, and kanamycin (100 IU) for 24 h to allow cell adhesion. Cells were subsequently treated with 5 or 15 mM glucose in the presence or absence of NAC (1 mM) for 24 h. Intracellular ATP levels were measured using a CellTiter-Glo Luminescent Cell Viability Assay kit (Promega, Madison, WI, USA), according to the manufacturer’s instructions. Luminescence was measured using a Glomax Multi Plus Detection System (Promega, Madison, WI, USA). The amount of ATP was calibrated by the number of cells.
Glomax multi plus detection system
Manufactured by Promega
Sourced in United States
The Glomax Multi Plus Detection System is a versatile luminescence detection platform designed for a wide range of applications. The system features a sensitive detector and specialized optics to accurately measure luminescent signals, providing precise quantification of various reporter assays.
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Lab products found in correlation
Caspase glo 3 7 assay kit, by Promega (2 mentions)
Bicinchoninic acid assay, by Thermo Fisher Scientific (2 mentions)
Celltiter glo luminescent cell viability assay kit, by Promega (1 mentions)
Lactate glo assay, by Promega (1 mentions)
Glucose uptake glo assay, by Promega (1 mentions)
Enduren live cell substrate, by Promega (1 mentions)
Celltiter glo, by Promega (1 mentions)
Gsh glo glutathione assay, by Promega (1 mentions)
Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Dual glo luciferase assay system, by Promega (1 mentions)
Pmirglo dual luciferase mirna target expression vector, by Promega (1 mentions)
Site directed gene mutagenesis kit, by Beyotime (1 mentions)
Pierce ripa lysis and extraction buffer, by Thermo Fisher Scientific (1 mentions)
11 protocols using glomax multi plus detection system
1
Glucose-Induced Cardiomyocyte ATP Modulation
2
Imatinib Cytotoxicity and Metabolism
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A total of 5×103 cells/well were seeded into 96-well plates. After each treatment with different concentrations of imatinib (0, 6.5 and 13 nM) for 72 h at 37°C, the culture medium was collected and cell viability and glucose and lactate concentrations were measured using the CellTiter-Glo 3D Cell Viability Assay (cat. no. G9681; Promega Corporation), Glucose Uptake-Glo Assay (cat. no. J1341; Promega Corporation) and the Lactate-Glo Assay (cat. no. J5021; Promega Corporation), respectively. All procedures were performed according to the manufacturer's protocols of each kit. Absorbance was measured using the GloMax Multi plus Detection System (Promega Corporation) to calculate the relative concentrations of lactate and glucose.
3
Fusion Assay Protocol for Dual Split Protein
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For the fusion assay, cell lines stably expressing DSP were utilized (see above), the stable cell line expressing dual split protein DSP8–11, were transfected with expression vectors of interest in quadruplicate. At 48 h post-transfection, 293CD4/DSP1–7 cells (2×104), a stable cell line expressing CD4 and DSP1–7, were co-cultured with transfected 293FT/DSP8–11 cells at 37°C in fresh medium containing membrane-permeable Enduren Live Cell Substrate (Promega). The RL activity was measured at 2hr after co-culture using GloMax-Multi Plus Detection System (Promega).
For designated experiments, the same batch of transfected 293FT/DSP8-11 cells were subjected to FACS analysis as described above. The RL activity readings were normalized to the respective MFI to compensate for the differential surface expression level of Env.
For DSP assay of cells after staining with Halo ligand, transfected cells were labeled for 15 min at 37°C with 1 µM of HaloTag TMR or Alexa Fluor 488 ligand. After labeling, cells were rinsed three times with 200 µl prewarmed DMEM/FBS and subsequently incubated at 37°C with 5% CO2 for 30 min. After the medium was replaced with fresh warm DMEM/FBS, images were captured using a microscope, then this sample was used directly for DSP assay.
For designated experiments, the same batch of transfected 293FT/DSP8-11 cells were subjected to FACS analysis as described above. The RL activity readings were normalized to the respective MFI to compensate for the differential surface expression level of Env.
For DSP assay of cells after staining with Halo ligand, transfected cells were labeled for 15 min at 37°C with 1 µM of HaloTag TMR or Alexa Fluor 488 ligand. After labeling, cells were rinsed three times with 200 µl prewarmed DMEM/FBS and subsequently incubated at 37°C with 5% CO2 for 30 min. After the medium was replaced with fresh warm DMEM/FBS, images were captured using a microscope, then this sample was used directly for DSP assay.
4
Cell Viability Assay for HTR-8/SVneo Cells
5
Quantifying GSH levels in HTR-8/SVneo cells
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The effect of DCVC on GSH levels was measured in HTR-8/SVneo cells by using the commercial GSH-Glo glutathione assay (Promega) following the manufacturer's protocol. This assay is based on glutathione S-transferase catalysis of a luciferin derivative to luciferin in the presence of GSH, coupled with luciferase to generate a luminescent signal. Briefly, cells were cultured at a density of 10 000 cells/well in a 96-well clear-bottom, white plate for 24 h and then treated with 10, 20, or 50 μM DCVC for 24 h. Treatment medium was removed, and then the cells were washed with PBS. Aliquots of 100 μl of prepared GSH-Glo reagent were added to each well. The plate was briefly placed on a plate shaker for 2 min for mixing. The plate was removed and incubated at room temperature for 30 min. Aliquots of 100 μl of reconstituted luciferin detection agent were then added to each well. The plate was again placed on a plate shaker for 2 min. The plate was removed from the shaker and incubated at room temperature for 15 min. Luminescence was read using a Glomax Multi Plus detection system (Promega).
6
Bcl-xL Modulation of Caspase-3/7 Activity
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A total of 1 × 104 cells (N134 parental cells and N134 expressing wt Bcl-xL, HA-Bcl-B/xL(BH4-loop) (construct #5), or RCASBP-HA-Bcl-xL/B(BH4-loop) (construct #6)) were seeded in 100 μL culture medium in a 96-well white wall plate. After overnight incubation, cells were treated with DMSO or 10 mM etoposide for 24 hours. Caspase-3 and -7 activities were measured using the Caspase-Glo 3/7 assay kit (Promega, G8090) according to manufacturer’s instructions. Luminescence was measured using a Glomax Multi Plus Detection System (Promega, Madison, WI, USA), and caspase 3/7 activities was normalized by the number of cells. Data represented 3 replicates per condition and were expressed as mean of fold change over N134 cells without etoposide ± SEM.
7
MMP2 3'-UTR Luciferase Assay Protocol
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The 647 bp of MMP2 3′-UTR containing the putative binding site for N-72 was cloned into pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega) using forward primer 5′-CgagctcCTCCACTGCCTTCGATACACC-3′ with a SacI site (lowercase) and reverse primer 5′-GCtctagaGAGACTCGGTAGGGACATGC-3′ with an XbaI site (lowercase). The mutation in the putative seed region was achieved using a Site-directed Gene Mutagenesis Kit (Beyotime, Haimen, China) and the following primers: 5′-CTTTCACAACCTTCTGTGGCTAGAAGAACCCTTGGAGCCAATGG-3′ (forward) and 5′-CCATTGGCTCCAAGGGTTCTTCTAGCCACAGAAGGTTGTGAAAG-3′ (reverse). 293T cells seeded on 24-well plates in DMEM (Hyclone) containing 10% FBS were co-transfected with either 50 nmol of N-72 mimics or NC and 200 ng of endo-free purified (Endo-Free Plasmid Midi Kit, Omega bio-tec, Guangzhou, China) wide-type or mutant plasmid using Lipofectamine 3000 Transfection Reagent (Invitrogen). The relative luciferase activities were determined at 48 h after transfection using the Dual Glo Luciferase Assay System (Promega) on GloMax Multi Plus Detection System (Promega).
8
Intracellular ATP Quantification by Luminescence
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The intracellular ATP level was measured using the CellTiter-Glo Luminescent Cell Viability Assay kit. Cells were cultured at 5 × 103 cells per well in a 96-well plate in RPMI-1640(DMEM) medium supplemented with 10% FBS, and allowed to grow overnight. Cells were given treatment with 2-DG (2.5 mmol/L), Rho123 (1.5 μg/ml) and the mixture of 2-DG (2.5 mmol/l) and Rho123 (1.5 μg/ml) for 6 h, respectively. The ATP level in cells was then determinate using a CellTiter-Glo Luminescent Cell Viability Assay kit according to the manufacturer’s protocol. Luminescence was measured using the Glomax Multi Plus Detection System (Promega, Madison, WI, USA). The luminescence signal is proportional to the amount of ATP as an index of cell number.
9
Caspase Activity Assay in Cardiomyocytes
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Caspase activity was detected using a Caspase-Glo® 3/7 assay kit (Promega, Madison, WI, USA). Briefly, cardiomyocytes were cultured at 1.5 × 105 cells per sample on either 15 kPa PAA gels or glass coverslips in DMEM medium supplemented with 5% FBS, 10 mM HEPES, and kanamycin (100 IU). After 24 h of culture to allow cell adhesion, cells were treated with either 5 or 15 mM glucose in the presence or absence of NAC (1 mM) for 24 h. As a positive control, cells were treated with staurosporine (1 μM) for 24 h to activate caspase-3. Caspase-3/7 activity was determined according to the manufacturer’s instructions. Luminescence was measured using a Glomax Multi Plus Detection System (Promega, Madison, WI, USA), and caspase 3/7 activity was calibrated by the number of cells.
10
PEP Activity Assay Protocol
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The PEP activity was assayed in a 110-μL reaction mixture containing 50 mM Tris-HCl, 150 mM ammonium sulfate, 0.02% Triton X-100, and various concentrations of the substrate. The reaction was started by adding the PEP solution to a final concentration of 0.25–2.0 μM. The reaction was performed at 25 °C, 50 μL of the reaction mixture was transferred to a white 96-well microtiter plate (Thermo Scientific 236105), and the fluorescence intensity was immediately measured by using a Promega GloMax®-Multi plus Detection System (Promega, Madison, WI) with excitation at 405 nm and emission at 495–505 nm.
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