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Method Overview

Measurements must be made in a plate reader, as the plate reader with a 96 well format is ideal as it provides a convenient format for multiple measurements. The methods are written from the perspective of using a 96 well format.

The first objective is to obtain your standard curves for fluorescence based on the sodium fluorescein reference material provided. Because the sodium fluorescein should fluoresce in the same way across different labs, measuring a standard curve of fluorescein values will allow us to calibrate the values obtained from your plate reader so that it can be compared with data from other labs. The standard curve must be obtained under EXACTLY the same instrument conditions that you will use when you do your cell based expression assays. This includes all settings that affect the amplitude of the signal collected: filters or monochromator settings; slit widths; gain settings; plates or cuvette type used; measurement from top or bottom (in plates); number of reads (integration time); orbital averaging (available in some plate readers). You may not know right now what settings will be suitable when you do your cell based assays. The objective is to fix the obvious settings now, such as monochromator/ filter, top or bottom reads. The key settings that affect sensitivity are slit width and/or gain. You MUST therefore collect now several standard curves under different sensitivity settings.

When doing cell based assays you should as far as possible settle on a single setting, but this may not be possible as it may not have sufficient dynamic range. By having a series of standard curves collected with different sensitivity you can use one (of a limited number) of the settings to obtain appropriate data (i.e. within range on the instrument). Because you will have a standard curve to match this setting, you can still transform into absolute units.

Recommended Filters: Excitation of 485nm, Emission 530/30 (or as close as possible to that range).

Plate Reader Protocol Details

We have provided detailed instructions, included background material, in the PDF below. We recommend that each team downloads this protocol when carrying out the experiments and copy any notes on those pages and in your lab notebook.

Required Data Forms

Each team is required to fill out the following data forms (Excel sheet, three online Forms).

Note: These forms will be posted by the end of May - please check back.