The total ATP levels were measured using the kinaseglo kit (Promega). Briefly, the membrane extracts were freeze-thawed and mixed with kinaseglo reagent. The bio-luminescence was measured using a luminometer plate reader (BioTek). The ATP levels were normalized to the amount of total protein in each membrane compartment.
Luminometer plate reader
Manufactured by Agilent Technologies
Sourced in Morocco
The Luminometer plate reader is a multipurpose instrument designed to measure luminescence in microplates. It provides accurate and reliable quantification of light-based signals, enabling the detection and analysis of a wide range of luminescent samples.
Automatically generated - may contain errors
Lab products found in correlation
Luciferase assay kit, by Promega (2 mentions)
White 96 well plate, by Corning (2 mentions)
Kinase glo kit, by Promega (1 mentions)
Caspase glo assay kit, by Promega (1 mentions)
Non silencing sirna, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Dual luciferase reporter system, by Promega (1 mentions)
Office scanner, by Epson (1 mentions)
Passive lysis buffer (plb), by Promega (1 mentions)
Luciferase assay system, by Promega (1 mentions)
7 protocols using luminometer plate reader
1
Quantifying Membrane ATP Levels
2
Caspase-3/7 Activity Measurement in Anticancer Treated Cells
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Cells were treated with anticancer agents for 24 h. Caspase-3/7 activity in cells was measured using a Caspase-Glo Assay Kit (Promega, Madison, WI, USA) with a luminometer plate reader (Bio-Tek).12 (link),17 (link)–21 (link) The relative caspase-3/7 activity was determined and normalised by cell viability, then the relative values were normalised by the value determined in untreated counterpart cells, set as 1 (X).
3
Trim28 siRNA Knockdown of Gal4-Mediated Transcription
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HEK293T cells (purchased from ATCC Catalog # CRL-11268) were transfected using Lipofectamine 2000 (Invitrogen) with pGL3-5XUAS Firefly luciferase reporter, a Gal4DBD effector and pRL Renilla luciferase plasmids. Total amount of DNA transfected was held constant by cotransfecting pCMV-MYC as needed. Cells were assayed with the Dual-Luciferase Reporter System (Promega) 24 hours after transfection. For small interfering RNA (siRNA) knockdown, 8 pmol of Trim28 siRNAs #1 (19779), #2 (19778) or non-silencing siRNA (Ambion) was transfected using Lipofectamine RNAiMax (Invitrogen). Cells were transfected with luciferase effectors and reporters 24 hours after siRNA transfection and luciferase was assayed after another 48 hours. For each luciferase assay, duplicate transfections and replicate lysates were measured for each condition (n = 4). Luminescence was quantified with a luminometer plate reader (BioTek). Firefly luciferase expression was normalized to Renilla to control for transfection efficiency. Fold repression was calculated compared to Gal4DBD. Lysates were analyzed by western blotting (not shown) to ensure consistent protein expression (protein loading was normalized to Renilla expression). Statistical analysis was performed using paired, two-tailed t-test.
4
Luciferase Assay for Adipocytes
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In vitro luciferase assays were performed using luciferase assay kits (Promega, Madison, WI) according to the manufacturer’s instructions. Remove culture medium from differentiated adipocytes and wash cells in PBS. Dispense an appropriate volume of 1X lysis reagent (Passive Lysis Buffer) into each culture well. Scrape attached cells from the wells, and transfer the cell lysates into white 96-well plate (Corning Inc., Tewksbury, MA) for detection of the bioluminescence signal using luminometer plate reader (BioTek Instruments, Inc., Winooski, VT). Use a reagent injector to dispense 100 μL of Luciferase assay buffer with substrate and 100 μL of Stop & Glo Reagent. And perform a 2-second pre-measurement delay, followed by a 10-second measurement period for each reporter assay. Luciferase activity data were normalized to protein content.
5
Luciferase Assay for Adipocytes
6
Colony Formation Assay Protocol
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After treatment, cells were trypsinized, counted, and 1000 or 5000 cells were plated in triplicate in 6-well plates containing complete RPMI medium and drug, as indicated. Cells were allowed to grow under standard conditions for at least 4 days until colonies were observed. For staining, 1 ml of crystal violet stain (0.05% crystal violet, 1% formalin, 1% methanol in PBS) was added to the cells. Cells were destained in deionized water, and images were taken using an Epson office scanner under film settings. To quantify stain, crystal violet was extracted using 1.5 ml of 1% SDS, followed by absorbance measurement at 612 nm wavelength light on a BioTek luminometer plate reader. Readings were also conducted in triplicate for each sample.
7
IFN-β Bioassay for Peptide-dsDNA Complexes
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IFN-β bioassay was carried out similarly to prior work55 (link). Supernatants were assayed for type I IFN production (IFN-β) using ISRE-L929 IFN reporter cells (Bio-ELISA system with IFN-β receptor coupled to a luciferase promoter). ISRE-L929 cells were grown to confluency in RPMI + BGS + Pen/Strep + l -glutamine and plated to a final concentration of 105 cells per well in a 96-well microplate. Fifty microliters of supernatant was incubated with ISRE-L929 cells for 7 h at 37 °C. An eight-point standard curve with twofold serial dilutions of known IFN-β concentrations (R&D Systems) was incubated with ISRE-L929 cells at the same time. ISRE-L929 cells are lysed overnight at −80 °C in Passive Lysis Buffer (Promega). Luciferase production in ISRE-L929 is quantified using a Luciferase Assay System (Promega) with a luminometer plate reader (Biotek). Luciferase production was converted to IFN-β production using the calibrated IFN-β standard curve. Peptide-dsDNA complexes that induce a TLR9-specific response produce a significantly larger amount of IFN-β from WT stimulation compared to TLR9KO stimulation (unpaired Student’s t-test).
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