2 replies to this topic

[Brightside]

Hi all

I have an EGFP-based reporter cell line for viral infection. The cells are easily infected with virus and the eGFP is highly expressed (as can be visualised by flourescence microscopy). Despite the appreciable difference in fluorescence between the infected and non-infected cells, I can not obtain a discernible difference using a plate-based flourometer. I have tried nearly everything I can think of - 12, 24, 96 and 384 wells; black, white and clear plates, shaking prior to reading, multiple reading points, ensuring that the flourimeter is working; growing the cells in phenol-red free media, washing the cells to remove FBS; etc etc but I have had no luck. The flourescence readings differ with different parameters (i.e. the readings are lower in the bigger wells) but there is no statistical difference between the + and the - (and I really aiming for fold-change differences).

So, my question is: has anyone detected eGFP flouresnce in live mammalian cells using a plate-based flourimeter? Can it be done? Or do I have to lyse the cells?

Any help or advice would be greatly appreciated!

Many thanks,
B

[virusfan]

Brightside, on 11 November 2010 - 05:49 AM, said:

Hi all

I have an EGFP-based reporter cell line for viral infection. The cells are easily infected with virus and the eGFP is highly expressed (as can be visualised by flourescence microscopy). Despite the appreciable difference in fluorescence between the infected and non-infected cells, I can not obtain a discernible difference using a plate-based flourometer. I have tried nearly everything I can think of - 12, 24, 96 and 384 wells; black, white and clear plates, shaking prior to reading, multiple reading points, ensuring that the flourimeter is working; growing the cells in phenol-red free media, washing the cells to remove FBS; etc etc but I have had no luck. The flourescence readings differ with different parameters (i.e. the readings are lower in the bigger wells) but there is no statistical difference between the + and the - (and I really aiming for fold-change differences).

So, my question is: has anyone detected eGFP flouresnce in live mammalian cells using a plate-based flourimeter? Can it be done? Or do I have to lyse the cells?

Any help or advice would be greatly appreciated!

Many thanks,
B

Since you mentioned that there was a difference in the fluorescence between infected and non-infected cells, can you take a tile image with a confocal microscope from several fields, and quantify the total fluorescence intensity with software? To do this, you need to use the same setting (gain, offset, etc.) for both infected and non-infected cells.

[genehunter]

The sensitivity with plate-reader is limited. More people use FACS to get average Fluorescence intensity, or immune precipitation or WB for EGFP protein.