Fragments of the ULBP1 promoter were amplified from HAP1 genomic DNA and inserted into the pGL3-Basic luciferase expression vector (Promega, Fitchburg, WI) between the KpnI and HindIII restriction sites. Mutant ULBP1 promoter fragments were ordered from IDT (Coralville, IA). The sequences of the ULBP1 promoter constructs can be found in Supplementary files 2–7 . ATF4-mutant HAP1 cells were plated at 10,000 cells/well in 96-well plates. 24 hr after plating, cells were co-transfected with the indicated ULBP1 promoter constructs (99 ng), pRK5-FLAG-ATF4 or control vector (199 ng), and renilla luciferase vector pRL-SV40 (1 ng). 24 hr post-transfection, cells were washed twice in PBS and lysed with 50 µl of passive lysis buffer (Promega #E1941). 15 µl of lysate was transferred to an opaque 96-well assay plate. 100 µl of D-Luciferin reagent (synthesized in-house) was added to the lysates and samples were immediately assessed for luminescence over a 10-s time period using an LMAX-II luminometer (Molecular Devices, Sunnyvale, CA).
Lmax 2 luminometer
Manufactured by Molecular Devices
Sourced in United States
The LMax II luminometer is a compact, high-performance instrument designed for sensitive luminescence detection. It utilizes a photomultiplier tube (PMT) to measure light output from a wide range of luminescence-based assays. The LMax II is capable of performing various luminescence measurements, including reporter gene assays, bioluminescence, and chemiluminescence. Its core function is to accurately quantify and analyze luminescent signals generated from experimental samples.
Automatically generated - may contain errors
Lab products found in correlation
Atp determination kit, by Thermo Fisher Scientific (2 mentions)
Pgl3 basic luciferase expression vector, by Promega (1 mentions)
Passive lysis buffer (plb), by Promega (1 mentions)
Reporter lysis buffer, by Promega (1 mentions)
Lipofectamine ltx transfection reagent, by Thermo Fisher Scientific (1 mentions)
Firefly luciferase assay kit, by Promega (1 mentions)
Nano glo hibit lytic assay system, by Promega (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Softmax pro software, by Molecular Devices (1 mentions)
Vialight plus kit, by Lonza (1 mentions)
Caspase glo 3 7 kit, by Promega (1 mentions)
Caspase glo 3 7 assay, by Promega (1 mentions)
11 protocols using lmax 2 luminometer
1
Transcriptional Regulation of ULBP1 by ATF4
2
Measuring Cytosolic Calcium Levels in Yeast
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A pYX212-based plasmid containing a functional apoaequorin gene (pAEQ) was transformed into yeast using URA3 gene as the selectable marker. This plasmid was a gift from Enzo Martegani (Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy). For analysis of basal cytosolic concentrations of Ca2+, cells containing pYX212-cytAEQ were inoculated in CSMD (–Ura –Leu) at OD600 = 0.1 and grown to mid log phase (OD600 ≈ 1.0). Ten A600 units were harvested and processed as described previously (Miseta et al., 1999 (link)). Luminometric analysis of the strains was carried out as kinetic (at 1-s intervals) and 20-s endpoint readings. An LMax II luminometer (Molecular Devices, Sunnyvale, CA) was used to collect aequorin light emission data. Data points are the average of triplicate determinations, and the error bars represent the range. The experiments were carried out three times independently, and a representative data set is presented in this study. The [Ca2+]cyt values were derived from luminometric units using a previously described equation (Allen et al., 1977 (link)).
3
SRF Reporter Gene Assays in NIH 3T3 Fibroblasts
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SRF reporter gene assays were performed as described previously (Copeland and Treisman [39 (link)]). Briefly, NIH 3T3 fibroblasts were seeded on 6-well plates at 125 000 cells/well 1 day prior to transfection. The cells were transfected using polyethylenimine transfection reagent (PEI) with the indicated expression plasmids (0.1ug) and the reporter constructs p3D.A.Luc (50 ng/well) and pMLVLacZ (250 ng/well). After 5 h, the cells were placed in low serum medium (0.5 % FBS in DMEM). Cells were harvested the next day and lysed in 1x Reporter Lysis buffer; luciferase assays were performed according to the supplied protocol (Promega) and read in an LMAXII Luminometer (Molecular Devices). The activation of luciferase was standardized to an SRF-VP16 control. A β-galactosidase (β-gal) assay is performed in parallel as a transfection efficiency control. The cell lysates are also subjected to a sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and immunoblotted to detect FMNL2 expression levels.
4
Transcriptional Regulation of Tyrosine Hydroxylase
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Human SH-SY5Y neuroblastoma cells were maintained at 37°C with 95% air and 5% CO2 in DMEM/F12 media supplemented with plus GlutaMax (Life Technologies). For transcription assays, cells were plated on Primaria 6-well plates, and Lipofectamine LTX transfection reagent (Life Technologies) was used to transfect 1.5 μg of a pGL4.20 luciferase reporter plasmid containing the 4.5kb upstream region of the rat Th promoter. At the time of transfection, cells were also treated with either saline or 100 μM of either TMPyP2 or TMPyP4. After 24 hours, cells were harvested and luciferase activity levels were measured using the firefly luciferase assay kit (Promega). Luminescence was measured with LMaxII luminometer (Molecular Devices). Luciferase activities are reported as the mean of at least three independent measurements with error bars representing the standard deviation.
5
Nanoluciferase-based cell lysis assay
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Twenty-four to forty-eight hours post-transfection, luminescence measurements were performed using the Nano-Glo HiBiT lytic assay system (Promega). Briefly, confluent cells were detached and lysed in a buffer containing the recombinant N-terminus of nanoluciferase (LgBiT) and nanoluciferase substrate furimazine in 96-well half-volume plates. Lysates were incubated for 10 min rotating, and luminescence intensities were recorded on a LMax II luminometer (Molecular Devices) at a one second integration time.
6
ATP Quantification in Mammalian Cells
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Cells were harvested by scraping into ice-cold PBS and then pelleted by centrifuging at 1000 r.p.m. for 5 minutes. Cells were then lysed by resuspending in urea and CHAPs lysis buffer [9M urea, 2% CHAPS in 30 mM Tris with 1× protease inhibitor cocktail (Roche)]. Samples were vortexed and briefly spun at 20,000 g. The ATP levels in fresh samples were determined using an ATP determination kit (Invitrogen) according to the manufacturer's instructions. Luminescence was recorded using an L Max II Luminometer (Molecular Devices) in a 96-well format. ATP concentration was normalised to the protein concentration that was measured by the Lowry-based detergent compatible (DC) protein assay (BioRad).
7
Luciferase Assay of KLF5 3'UTR
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SNU-5 cells were transfected with 100 nM WT-KLF5-3′UTR or MT-KLF5-3′UTR plasmid, as well as with or without 100 nM miR-153 mimic. The luciferase activity was then measured at 48 h after transfection using the Dual-Luciferase Reporter Assay System (Promega Corporation) on an Lmax II Luminometer (Molecular Devices, LLC, Sunnyvale, CA, USA). The firefly luciferase activity was then normalized to the Renilla luciferase activity.
8
Quantifying Cellular ATP Levels in iPSC-Derived MNs
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Total cellular levels of ATP of iPSC-derived MNs were measured using ViaLight Plus kit (Lonza) according to manufacturer’s instruction. Dissociated 20,000 cells were plated in each well of Matrigel-coated 96-well plates. On the day of measurement, cells were first washed with PBS and lysed for 20 min in cell lysis reagent on a shaker to extract ATP. The cell lysates were then transferred to a 96-well clear bottom white polystyrene microplate (Corning) and ATP monitoring reagent plus was added to generate luminescent signal. After 1–2 min, the plate was placed in LMax II luminometer (Molecular Devices) and the ATP levels were measured with SoftMax Pro software (Molecular Devices).
9
Intracellular ATP Quantification in Neurons
10
Caspase Activity Measurement in Infected Cells
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The caspase assay was performed using the Caspase-glo 3/7 kit (Promega, USA) according to the manufacturer protocol. Vero cells were plated in 96 well plates 24 h before infection. The cells were then infected with the clones exhibiting complete suppression at an MOI of 0.05. At 2dpi, the cells were incubated with Caspase-glo reagent for 1 h in the dark at room temperature. The caspase activity was measured by detecting the luminescence using LMAX-2 luminometer (Molecular Devices).
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