We don't have a 96well TEER measurement capability, but I wanted to make sure that the tight junctions are good prior to experimentation.
Can lucifer yellow be used prior to the experiment to assess barrier function?
I was the planning to stress the cells, and re-assess the barrier function again with lucifer yellow.
Thank you for your help.
As far as I know its fairly stable in aquatic solutions, so it probably works.
If you determine that the LY is not toxic to your cells in your experimental conditions (MTT test ....) and you show that the LY does not alter the permeability of a positive control (if you have one! !!). Knowing that Ly is water soluble, I think so!
If I understand your experimental need correctly, you would like to test the barrier function with LY at zero time of the experiment, and then test it again after stressing the cells. I think the answer is yes. I suppose you are taking live cell images for penetration of of LY to the paracellular space. I think you can do it in two different ways. In the first, add LY to top and take images after washing to see if any fluorescence present between cells. Obviously, if the junctions are tight you will see any fluorescence, but you will it if junctions are leaky. You can do the same after stressing the cells to compare if stress has opened the junctions. If you are using a confocal microscopy, you can take Z-stack image from to to bottom, and view the Z-section image, and therefore, you do not have to wash the LY off. You will see bright fluorescence above the cells if the junctions are intact.
In the second approach, you can add LY to top before stress and take time-lapsed z-stack images before and after stress without removing LY. Hope this helps.
I've always just measured the movement of the LY from the cell insert (across the cell monolayer) into the collection well using a fluorescent plate reader.
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