I am currently doing cell viability test using fluorescence reading in microplate reader. Someone told me to use black 96well plate instead of completely transparent one for better readout. I checked a bit, seems most people say so. I understand the crosstalk issue when two staining agents are tested together; their emission/excitation wavelength will cross each other. However, the crosstalk taking place between two adjacent wells is unclear to me. Also, still some labmates who are currently doing fluorescence reading told me using the completely transparent well plate is fine. That made me very puzzled. Could anyone help me to resolve this? Is it necessary to use black one (plz tell me also in which condition transparent well is validated for Fluo-reading) . Much thanks!
Hi Kenny, I don't think it's a problem using a transparent plate. It depends on the instrument setting. For some fluorescence reader, there are multiple choices for you to choose, for example, a transparent one or a black one. The instrument will thus adjust its setting based on what kind of plate you use.
Dear Kenny
About the crosstalk or interference of two fluorescent agents having different excitation wavelength. if two fluorscent dyes are in the same sample and your reading at one of their excitation wavelength, that doesn't mean that one will not give you a reading and the other will. They will both still emit fluorescence regardless; except if there excitation wavelength are far apart or their interference is at minimal (like in this image attached). That is known, but If the dyes are separated into two wells then you already know which well has which dye and which wavelength/well(s) you are going to work with in each reading. However, the crosstalk between wells comes from that the emitted light from the sample is like a sphere it emits 360 degrees. So if the walls of the wells are transparent (black wells won't have that problem) one well would interfere (through diffraction and reflection of light) with the reading for that other well. Although it is minimal but can make a difference especially if the dye is bright in nature. That is why it is best to use one dye for one plate at a time so the cross talk from well to well is minimal.
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I hope I helped
@HAN ZHANG. Hi Han, I redo again for fluorescence study using CELLROX to read ROS in Hacat cell lines. I found that even blank control (means only cells and PBS before reading) can have very high fluorescence readout. I doubt that cells themselves could interfere with the fluorescence. subsequently, I used microscope to observe the actual staining status in each well (I was using trans 96wellplate); blank well didn't give any staining under microscope. That just further let me suspect the cells' interference. What do you think? also, I observed a bit orange background under microscope, does that mean I did not wash completely before reading?
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