HEK293 cells (ATCC CRL-1573.3) were cultured in growth media (DMEM, 10% heat inactivated fetal bovine serum, penicillin (100units/mL), streptomycin (100 μg/mL) and L-glutamine (290 μg/mL)) and used between passages 5–25. For GPCR heterologous expression and functional assays, cells were transfected (Lipofectamine, 2000) at 80% confluency approximately 16 h after seeding on T-25 culture flasks with a 1:1 ratio of Sm.5HTRL and the pGloSensor 22-F plasmid (Promega). Sm.5HTRL was codon optimized for human expression and subcloned into the pcDNA3.1(−) mammalian expression vector. Sm.5HTRL (GenBank accession, KX150867) is a longer form of the Sm.5HTR originally reported by Patocka et al. (Patocka et al., 2014 (link)). The following day, cells were trypsinized, centrifuged (300 g/5 min), resuspended in DMEM supplemented with 1% dialyzed FBS (Gibco) and plated in 96 well, solid white plates (Corning, cat # 3917). After overnight culture to allow adherence, media was exchanged for assay buffer (HBSS supplemented with 0.1% BSA, 20 mM HEPES (pH 7.4), and GloSensor reagent (Promega)). cAMP-luminescence assays were performed in the presence of phosphodiesterase inhibitor (IBMX, 200 μM) using a GloMax®-Multi Detection System plate reader (Promega).
Glomax multi detection system plate reader
Manufactured by Promega
Sourced in United Kingdom, United States
The GloMax®-Multi Detection System is a microplate reader that measures luminescent, fluorescent, and absorbance signals. It is designed to perform a variety of assays, including cell-based, biochemical, and molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Pglosensor 22 f plasmid, by Promega (2 mentions)
Glosensor reagent, by Promega (2 mentions)
Dual luciferase reporter assay system, by Promega (2 mentions)
96 multiwell black plate, by Corning (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
One glo luciferase assay system, by Promega (1 mentions)
Pgl4.30 luc2p nfat re hygro, by Promega (1 mentions)
Pierce ldh cytotoxicity assay kit, by Thermo Fisher Scientific (1 mentions)
Celltiter 96 aqueous non radioactive cell proliferation assay, by Promega (1 mentions)
Pierce firefly luciferase glow assay kit, by Thermo Fisher Scientific (1 mentions)
Hek293 cell, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Caspase glo 3 7 assay kit, by Promega (1 mentions)
Celltiter glo assay, by Promega (1 mentions)
Fluo 4 nw calcium assay kit, by Thermo Fisher Scientific (1 mentions)
Fitc dextran, by Promega (1 mentions)
Fitc dextran, by Merck Group (1 mentions)
Glo lysis buffer, by Promega (1 mentions)
Celltiter glo luminescent cell viability assay reagent, by Promega (1 mentions)
19 protocols using glomax multi detection system plate reader
1
Heterologous Expression of Sm.5HTR_L in HEK293 Cells
2
Transcriptional Luciferase Assay for 5HT2B Receptor
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The Ca2+ sensitive transcriptional luciferase reporter pGL4.30[luc2P/NFAT-RE/Hygro] (Promega, E8481) was transiently transfected into the 5HT2BR stable cell line. Briefly, 3 × 106 cells were plated in a Nunc Cell Culture Treated flask (25 cm2, ThermoFisher) and transfected with lipofectamine 2000 (ThermoFisher) plus 1 µg of plasmid DNA according to the manufacturer’s protocol. The following day, culture media was replaced with induction media (DMEM + 10% dialyzed FBS supplemented with 1 µg/ml doxycycline), and 24 h later cells were re-plated into 96 well, solid white plates (Costar, 3917). After allowing 3 h for cells to adhere, drugs were added at 20× concentration. For antagonist experiments, cells were incubated with 5HT2B R antagonists for 2 h prior to subsequent addition of agonist. After 18 h culture in the presence of agonist, plates were assayed using the ONE-Glo Luciferase Assay System (Promega, E6120) and read on a GloMax-Multi Detection System plate reader (Promega).
3
Bacterial Culture and Quantification
4
LDH Cytotoxicity Assay for Stretch Injury
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Media samples were taken from each well of flex plate before stretch, 4hr and 24hr following injury. Samples were tested for LDH levels [32 (link)] using the Pierce LDH Cytotoxicity Assay Kit (Thermo-Fisher). Results were read using a Promega Glomax Multi Detection System Plate Reader using 490 nm and 690 nm filters.
5
Dual-Luciferase Assay for PARP1 and Zta
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Luciferase assay was performed using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions. HEK293 Cells were transfected with pRL, LucZ, and constructs expressing either His-PARP1 or FLAG-Zta using Lipofectamine 2000 according to the manufacturer’s protocol. After 72 h, cells were lysed for 15 min at room temperature in the supplied Passive Lysis Buffer and transferred into a 96-well plate for luciferase assay. LARII and Stop & Glo Reagents were dispensed into wells via automated injectors. Luminescence was measured using the Promega GloMax Multi Detection System plate reader.
6
Cell Viability Assay with MTS Reagent
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Cell viability was assessed using the CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay (Promega). 100μL of the MTS reagent was added to each 1 mL of media in flex plates and were returned to the incubator for 100 minutes. Results were read using 100μL of solution in a 96 well plate using a Promega Glomax Multi Detection System Plate Reader using 490 nm and 690 nm filters.
7
Luciferase Expression Assay in HEK293 Cells
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HEK293 cells were seeded into 96 well tissue culture plates at 1x105 cells per well and 24 hours later transduced with the treatment and control AAV samples at an MOI of 10,000. Three days later, the cells were harvested and lysed with the 2X Cell Lysis buffer provided in the kit. Lysates were analyzed for luciferase expression using the Pierce™ Firefly Luciferase Glow Assay Kit (ThermoFisher, cat. no. 16176) and the GloMax®-Multi Detection System plate reader (Promega) as per the manufacturer’s instructions
8
Streptococcus culture and quantification
9
MTT Assay for RPE Cell Viability
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The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis is an established assay for evaluating metabolic activity of cells, and can be useful for the measurement of cell viability. MTT analysis was accomplished as previously reported for determining the metabolic activity of RPE cells.13 (link) The medium was removed, the cells were washed with PBS, 1000 mL/well MTT solution was added, and the cells were incubated at 37 °C for 1 hour. The formazan crystals were dissolved after the administration of DMSO (1000 mL/well). Absorption was evaluated by a scanning spectrophotometer (Promega Glomax MultiDetection System Plate reader, USA) at 560 nm. The procedures were carried out 3 times. Treatment-naive RPE of the same passage served as the control.
10
Heterologous GPCR Expression in HEK-293 Cells
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HEK-293 cells (ATCC CRL-1573.3) were cultured in growth media [DMEM (Gibco), 10% heat inactivated fetal bovine serum (Gibco), penicillin (100units/mL), streptomycin (100μg/mL) and L-glutamine (290μg/mL)] and used for assays between passages 5 and 25. For cestode GPCR heterologous expression assays, cells were transfected (Lipofectamine 2000, Invitrogen) at 80% confluency approximately 16 hours after seeding within T-25 culture flasks with a 1:1 ratio of human codon optimized cestode GPCR cDNA (subcloned into a pcDNA3.1(-) mammalian expression vector) and cDNA encoding the pGloSensor 22-F plasmid (Promega). The following day, cells were trypsinized, centrifuged (300g/5min), resuspended in DMEM supplemented with 1% dialyzed FBS (Gibco) and plated in 96 well, solid white plates (Corning, cat # 3917). After overnight culture to allow adherence, media was exchanged for assay buffer (HBSS supplemented with 0.1% BSA, 20mM HEPES (pH 7.4), and GloSensor reagent (Promega). cAMP-luminescence assays were performed following addition of a phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine, IBMX; 200μM) using a GloMax-Multi Detection System plate reader (Promega) as described previously [24 (link)].
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