When it comes to purchasing a microplate reader for fluorescence detection, the most common question is whether to choose a monochromator-based reader or filter-based reader. In this blog, we’ll discuss how both types of plate readers work and factors to consider when determining the best plate reader for your need.
How do monochromator-based plate readers work?
Monochromators work by taking a light source and splitting the light to focus a particular wavelength on the sample. During excitation, the light passes through a narrow slit, directed by a series of mirrors and diffraction grating and then passes through a second narrow slit prior to reaching the sample. This ensures the desired wavelength is selected to excite the fluorophore. Once the fluorophore is excited, it emits light at a different, longer wavelength. This emission light is captured by another series of mirrors, grating and slits to limit the emission to a desired wavelength, which then enters a detector for signal readout.
Bioluminescent reporter assays are an excellent choice for analyzing gene regulation because they provide higher sensitivity, wider dynamic range and better signal-to-background ratios compared to colorimetric or fluorescent assays. In a typical genetic reporter assay, cells are transfected with a vector that contains the sequence of interest cloned upstream of a reporter gene, and the reporter activity is used to determine how the target sequence influences gene expression under experimental conditions. A second control reporter encoded on the same or a different plasmid is an essential internal control. The secondary reporter is used to normalize the data and compensate for variability caused by differences in cell number, lysis efficiency, cell viability, transfection efficiency, temperature, and measurement time.
For genetic reporter assays, using a secondary control vector with a weak promoter like PGK or TK to ensures that the control does not interfere with activation of your primary reporter vector. Transfection of high amounts of the control plasmid or putting the control reporter under control of a strong promoter like CMV or SV40 often leads to transcriptional squelching or other interference with the experimental promoter (i.e., trans effects). Reporter assays can also be used to quantitatively evaluate microRNA activity by inserting miRNA target sites downstream or 3´ of the reporter gene. For example, the pmirGLO Dual-Luciferase miRNA Target Expression Vector is based on dual-luciferase technology, with firefly luciferase as the primary reporter to monitor mRNA regulation and Renilla luciferase as a control reporter for normalization.
Here in Technical Services we often talk with researchers who are just starting their project and looking for advice on designing their genetic reporter vector. They have questions like:
How much of the upstream promoter region should be included in the vector?
How many copies of a response element will be needed to provide a good response?
Does the location of the element or surrounding sequence alter gene regulation?
Each luminescent assay plate represents precious time, effort and resources. Did you know that there are three things about your detection instrument that can impact how much useful information you get from each plate? 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.
Based on the Illuminations article by Dr. Terry Riss, from our Cellular Analysis group.
Choosing the most appropriate cell health assay for your experiment can be difficult. There are several factors to consider when choosing an assay: the question you are asking, the nature of your sample, the number of samples being tested, the required sensitivity, the nature of the sample, the plates and plate readers and the reagent costs.
What question are you asking?
The first, and perhaps most important factor to consider, is the question you need answered. What do you want to know at the end of the experiment? There are cell health assays available that specifically detect the number of living cells, the number of dead cells, and for assessing stress response mechanisms or pathways that may lead to cell death. Matching the assay endpoint to the information you need is vital to choosing the appropriate cell health assay. Continue reading “Choosing the Right Cell Health Assay”
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