How do you choose the best luciferase vector for your experiment?
To help you, Promega has developed a tool called the "luciferase reporter vector selector" You will be able to select your vector by choosing the type of promoter, the application that interests you, the selection marker, or the type of luciferase.
What is multiplexing?
Multiplexing different assays provides a set of data from the same sample. The main advantage is to have a better understanding of the event you are measuring in the context of another parameter, thus minimizing misinterpretation. The second advantage is that it allows you to standardize your results. To multiplex several assays, it is necessary that the signals of the various assays are spectrally or temporally distinct, the chemistries must be compatible, and be usable on the same well while being easily separated.
How do I authenticate my cell line?
Cell line authentication need not be daunting: identification can be achieved by genetic profiling using polymorphic STRs (shorts tandem repeats). ATCC issued standards that recommend the use of at least 8 STRs loci for identification. The GenePrint10 kit contains 10 loci (TH01, TPOX, vWA, Amelogenin, CSF1PO, D16S539, D7S820, D13S317, D5S818 and D21S11) and has the power to confirm whether your cell line is the one you think. This kit can be used on all sequencers Applied Biosystems.
What are common issues that could hurt my luminescent assay results?
Instruments with poor sensitivity may cause you to miss low-level samples that could be the “hit” you are looking for. Instruments with a narrow detection range limit the accuracy or reproducibility you needed to repeat your work. Finally, instruments that let the signal from bright wells spill into adjacent wells allow crosstalk to occur and skew experimental results, costing you time and leading to failed or repeated experiments.
How do I measure viability on 3D cells?
CellTiter-Glo 3D is a bioluminescent assay that measures the level of ATP as an indicator of viability, thus making it possible to quantify cell viability within 3D spheroids. CellTiter-Glo 3D works with a single reagent in liquid form and generates a luminescent readout that is much more sensitive than colorimetric or fluorescence-based methods. The assay reagent penetrates large spheroids and has increased lytic capacity—allowing more accurate determination of viability compared to other assay methods.
How do you quickly and efficiently measure your level of ROS?
ROS - "reactive oxygen species" produced by cells are molecule regulators involved in cellular signaling pathways. An excess of ROS can also induce cell death. The H₂O₂ level in a cell can reflect the general level of ROS in a cell. In the ROS-Glo H₂O₂ assay™ kit, A derivatized luciferin substrate is incubated with sample and reacts directly with H₂O₂ to generate a luciferin precursor. Addition of ROS-Glo™ Detection Solution converts the precursor to luciferin and provides Ultra-Glo™ Recombinant Luciferase to produce light signal that is proportional to the level of H₂O₂ present in the sample. With these assays, it is important to include multiple controls such as culture alone (which can contain or convert H₂O₂), with cells and a control positive as cells treated with menadione or pyrogallol.
Which plates do you use use for your fluorescence and luminescence measurements?
In experiments involving fluorescence and luminescence measurements, a question may arise about which plate will allow for the best signal with low background noise. For luminescence, we recommend white opaque plates and for fluorescence measurements, we recommend opaque plates black. Using white plates to measure fluorescence will reduce the signal to background ratio but will give usable results. While using black plates for luminescence measurements will imply a decrease in the background noise but strong signal reduction (15 to 20 fold difference; white vs black). So, for experiments combining both a measure of fluorescence and luminescence, we recommend the use of white plates.
What additional factors should I consider when choosing a cell viability assay?
Among the many factors to consider when choosing a cell-based assay, the primary concern for many researchers is the ease of use. When choosing an assay, the time required for reagent preparation and the total length of time necessary to develop a signal from the assay chemistry should be considered. Other factors to consider include sensitivity of detection, reagent stability and more. Check out the link below to learn more!
What is BRET?
BRET stands for Bioluminescence Resonance Energy Tranfer. This technique measures an interaction between two proteins by donor energy transfer bioluminescent fluorescent acceptor when the two proteins interact. BRET allows a co-location in the cell of a PPI interaction.
How do I develop a test to study protein-protein interactions with NanoBRET?
Promega scientist, Danette Daniels, explains how to set up a NanoBRET™ experiment, letting you fully explore protein interactions inside cells. Just click on the link below