I want to measure the fluorescence of a fluorescent protein in a 96-well plate in a plate reader (like Omega and Flexstation).
I read that the measure of fluorescence varies depending in what well is the fluorophore in (you obtain a different value for the same concentration and volume if you put it in the peripheral wells or in the central wells) and that the fluorescence of the neighbor wells can affect too.
So I want to know if you can give me an advice on how I put my samples ( I was thinking to fill the peripheral wells with my blank (buffer) and for reducing the neighboring well interference, I'll keep the wells empty around sample well).
Dear Proffessor,
Unfortunately, I have never well plates in measuring fluorescent samples.
Yours,
K. Fujimoto
If you have a doubt about your instrument, then the best start is to offer one plate and make a test experiment with any fluorophore (e.g. fluorescein), if you see a problem, consult it with your supplier.
In general, the 96-well plates should be less prone to the effect of adjacent cells illumination than the 384-well plates, it may depend on the quality of your instrument.
In my experience, the signal is non-linearly dependent on the volume in the cell, which may be critical. Good automatic pipette has typically accuracy of 1-2%, but in lower part of its range, it may exceed 5%. The problem can be even worse, when an unexperienced user loads the well with 8-channel pipette. Also the pipetting robots have typically the accuracy worse than 5%, especially for small volumes.
I have measured fluorescence in 96 well plate. In general, I used to avoid the peripheral wells and used to keep same solutions in two or three adjacent wells. Finally, I used to take the average of those data.
The only effect that is critical regarding edge wells, (called "edge effect") is slow evapration of the liquid inside the well (over hours, normally). The edge wells tend to evaporate faster, especially if you use a lid together with the plate. Evaporation leads to concentration change and chages the well's liquid level, therefore leading to a signal change.
Regarding neighboring wells, there is a performance parameter calld "cross talk", which is characteristic for the respective reader. I would say, any plate reader should be able to read 96well plates without considerable crosstalk. To test your for crosstalk, you might fill a well with strongly fluorescent solution and the wells around with solvent only. then measure all wells and compare the intensitiy of the direct enighbor wells to the indirect neighbor wells. If they give the same (small) intensity, you don't have a crosstalk problem.
For avoiding crosstal generally, ensure that neighboring wells give similar intensities, that is don't place a blank well neighboring a high concentation well.
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Physical Phenomena
- Luminescence
- Fluorescence