Our laboratory is not equipped with a flow cytometer. Is it possible to stain cell suspensions (i.e. from thymus) in 96 well plates with multiple fluorochrome conjugated antibodies and then read the plate in a fluorescent microplate reader for fast analysis of a large number of samples in order to estimate cell subpopulations?
Even if you could, and I don't think it'll work the way you think.
In flow the excitation lasers have a specific wavelength, I think the plate readers just uses white light. Unless you use fluorochromes that have no spill over in the emission wavelength, I don't see how you can account for false negatives.
Also, all you will get is a MFI right? There is no way to differentiate - versus + populations. And without the ability to do that... you can have higher MFI despite having a lower percent of positive cells.
Now... if you have the ability to take pictures... you might be able to train a program to do a count.
You'll need a lot of cells and a lot of label to make this work. Way more than you would for flow cytometry. It will be expensive.
And you miss out on the beauty of flow cytometry -- the rich data that tells you what's going on -- cell-by-cell information linked to size. How are you going to demonstrate that your fluorescence is not coming from some non-specific aggregate? Perhaps you can devise controls that make this useful as a first-pass screening, to shrink the sample set and confirm later by other methods. But I wouldn't count on being able to convince a peer reviewer of this as a primary means of generating evidence.
Dear Ioannis Roussos ,
There are lots of limitation you are going to face upon replicating effort of flow cytometry results in microplate reader including Laser variability, less sensitivity & specificity , non-specific fluorescent emission etc.
Best regards,
Babu M.
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