Hello,
I am doing a neuroprotective in vitro study on shsy5y cell culture. By using live/dead assay, it will stain live cells as blue, and dead cells as red. I used fluorescence microplate reader to detact the total fluorescence of blue and red. It gives me promising result in the experimental group compared to the control(more live than dead). However, when I count the cells by my bare eyes using fluorescence microscope, the live and dead cells in experimental group and control group look similar.
I am wondering why microplate reader can detect such a contrast difference, but it seems indifference by bare eyes?
Does everyone who use microplate reader also experience?
I have no experience but nevertheless a suggestion: If you have the chance you should analyze your cells by flow cytometry. One explanation could be an increase of injured (dying) cells in your experimental group. Flow cytometry will show you whether this is the case.
Hi,
Uwe may be right, and you must consider that the microplate reader reads both colors separately, whereas with your microscope, you will probably be using two excitation/emission filters and be looking at both colors at the same time, which means that the blue will be in all cells, whether live or dead, as it can permeate the cell membrane, and the red will be in dead or dying cells with damaged membranes. So, in effect, you will be seeing red superimposed on top of the blue, and this may not be as easy to differentiate if the cells are for the most part still dying and the red portion is thus weaker than the blue in the same cell.
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