Cells were plated as described for 2D or 3D culture, and then treated with compounds at the indicated concentration for 48 hours. To quantitate cellular ATP as a measure of viability, 50 µL of CellTiter GLO reagent (Promega) was added to each well. The plates were orbitally shaken for 15 minutes at room temperature to facilitate lysis. In addition, spheroids were triturated six times to promote complete lysis. Well contents were transferred to a white-bottom 96-well plate and read on a Bio-Tek luminescence plate reader.
Luminescence plate reader
Manufactured by Agilent Technologies
Sourced in United States
The Luminescence plate reader is a laboratory instrument designed to measure the luminescence of samples in a microplate format. It is capable of detecting and quantifying various types of luminescent signals, such as bioluminescence, chemiluminescence, and fluorescence.
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Lab products found in correlation
6 protocols using luminescence plate reader
1
Cell Viability Measurement via ATP
2
Apoptosis Induction in K9 Cell Lines
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K9TCC and K9OSA cell lines (1.5×106 cells) were grown in 10 cm petri dishes in complete media for 24 hours. After plating the cells, cells were treated with DOX (1 μM) and AD198 (1 μM) for 24 hours in serum-free media. After treatment, cells were lysed in ice-cold RIPA buffer as mentioned in the “Western blot analysis” section. Activities of caspases 3 and 7 were detected using the Caspase-Glo 3/7 Assay (Promega Corporation). Cell lysates containing 30 μg of proteins were incubated with a proluminescent substrate specific for caspase-3/7 at a 1:1 ratio in a 96-well plate at room temperature for 1 hour. The luminescence values for each treatment were measured with a luminescence plate reader (Bio-Tek Instruments, Inc.) and normalized to control groups.
3
Maslinic Acid Induces Apoptosis in Neuroblastoma
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Neuroblastoma SHSY-5Y cells were cultured under given conditions in 96-well plates with a density of 2 × 105 cells/mL. Afterwards, maslinic treatment (0, 10, 40 and 80 μM) was initiated in terms of positive controls and negative controls were only treated with 0.1% DMSO for 24 h. Caspase-Glo® assay (Promega, Madison, USA) was implemented according to the manufacturer’s protocol to estimate the activity of Caspase (3 and 9). Finally, luminescence was determined with a luminescence plate reader (BioTek, United States).
4
Cellular Bioenergetics of Daudi and PBMC
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Cellular ATP content was analyzed to determine the bioenergetics of the Daudi and normal PBMC’s. Cells subjected to treatment of hypoxia, with and without treatment of Mito-CP, were seeded, and intracellular ATP levels were determined using a bioluminescence-based assay as per the manufacturer’s protocol (Molecular Probes, Eugene, OR, USA). Luminescence were measured by luminescence plate reader (BioTek Instruments, Winooski, VT, USA).
5
Quantifying AIBP Binding to TLR4 and APOA1
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To assess AIBP-TLR4 binding, 96-well plates were coated with 5 µg/ml of eTLR4, washed three times with PBS containing 0.05% Tween-20, blocked with PBS containing 1% BSA, and incubated with wtAIBP or mutAIBP, followed by 2 µg/ml of a biotinylated BE-1 anti-AIBP mAb. To assess AIBP-APOA1 binding, plates were coated with BSA, wtAIBP, or mutAIBP; washed; blocked; and incubated with 5 µg/ml of human APOA1 (a gift from Dmitri Sviridov, Baker Heart and Diabetes Institute, Melbourne, Australia), followed by a biotinylated anti-APOA1 antibody (Academy Bio-Medical Company; RRID:AB_1238781). In both assays, neutravidin-AP was added and incubated for 45 min at room temperature, followed by LumiPhos 530 (Lumigen) for 90 min, and luminescence was measured using a luminescence plate reader (BioTek).
6
Metformin's Impact on Caspase-3/7 Activity
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To determine the activity of caspase-3 and -7 the cells were seeded in 96 well-plates (TPP, Faust Lab Science GmbH) with a cell density of 7,500 cells/well. After an adherence period of 12 h the cells were incubated with 1 mM metformin for 48 or 96 h. The caspase-3 and -7 activity was measured after exposure to the Caspase-Glo® 3/7 Assay (Caspase-Glo® 3/7 Assay, Promega) in accordance with the manufacturer`s instructions for cells cultured in a 96 well-plate after 2 h. The luminescence was measured with a luminescence plate reader (BioTek Instruments GmbH) and normalized to the untreated controls [54 (link)].
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