Fluorsence Problem

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Kelly M.
Kelly M.'s picture
Fluorsence Problem

I'm trying to do a serial dilution of Alexa 488 in a 96 well plate to create a concentraion curve (hopefully). I've done the experiment twice now, and the valuse I get from the plate reader are completely opposite from what we are expecting.  For example, my most concentraited sample gives the lowest readings and my least concentraited gives the highest. My blank (PBS) is also giving a very high reading.
To answer the obvious, I have the wavelength correctly set.  The plate is in the correct orientation. I've done my absolute best to minimize light exposure.  All dilutions are done in/with PBS.  We are using a black side, clear bottom 96 well plate with a volume of 200 uL.  We've read from the top and the bottom with no noticable difference in results. We've changed the sensitivity on the scan with no real results.

Any thoughts on why I keep getting non-sensical values or how you'd go about creating a known curve for Alexa concentrations?

The FFM
The FFM's picture
Things to check (please dont

Things to check (please dont be insulted by some that may appear extremely obvious  - but I have to ask to eliminate them)

Is everyone who uses the machine getting strange results with all flurophores - not just Alexa 488

Is the machine properly calibrated for each dye? 
Is the filter wheel stuck and its not actually using the correct filters even though the computer says that it is? 

Has someone "borrowed" the filters from the machine, or put the wrong ones back in without telling you?

Are you sure that the machine set to detect fluorescence and not absorbance or chemiluminescence?

When you say you have the wavelength set correctly do you mean BOTH the exciatation and emission wavelengths?

My initial instinct was that you accidentally had something set to read absorbance at 488 rather than exciting at 488 and detecting emmission at 525ish, but if your PBS control is high too that would apparently eliminate that possibility

Kelly M.
Kelly M.'s picture
I don't know if everyone who

I don't know if everyone who uses the machine is getting odd results.  One of the PhD's using it says it is working just fine for him (although i failed to ask at what wavelength/emission... and have a note to find him to ask).

There are no error messages from the machine saying the filter wheel is stuck. 

No one has opened the machine or borrowed the filters to my knowledge or to the knowledge of the person who owns the plate reader.

I am positive the software is set for fluorescence and not absorbance... this was one of the first things we thought to check.

Both excitation and emission wavelengths are set correctly.

I purchased Sodium Fluorescein and ran  a fluorescence test that was recommended by BioTek.  Using PBS (and distilled water) are giving values similar to what I get when I read Sodium Fluorescein (i can send values if you'd like).  The corners test passes, the linearity test almost passed (passed is >/= 0.95... we got 0.947...) and the sensitivity test failed.

Having talked to BioTek, they seem to think there is a contaminant that is autofluorescing and causing a problem but when the optics test passes and others are having it work for them, I'm not sure I can agree with that thought process...

Any other ideas?

victorius
victorius's picture
I would try and read the same

I would try and read the same plate in a different reader to rule out that the reader is the problem.

Kelly M.
Kelly M.'s picture
Thanks for all your help.

Thanks for all your help.  After much searching, I was able to find another fluorescence plate reader.  I applied the appropriate setting to the software and the plate read with levels I would expect (decreasing as the concentration decreases).   I took the same plate I got "good" readings on and re-read it on the plate reader that was giving me fits and got non-sensical numbers again (blanks reading as the same level as samples).  The owner of that plate reader still doesn't belive its the source of the problem though.... cest la vie.

jackiemorris01
jackiemorris01's picture
Hi Kelly,

Hi Kelly,
At the risk of asking the obvious.... have you asked the 'poor' plate reader owner (if you see what I mean) to run your test himself and then compare it to the same test on the 'good' plate reader. If it is his equipement producing 'poor' results then it may stop someone else getting poor data as a result of using inadequate equipement...and even more importantly may make the other person realise that they can't just dismiss the problem without checking it. Isn't that what research is all about?
Jackie

Kelly M.
Kelly M.'s picture
Jackie,

Jackie,
I did offer that when we were trying to solve the problem and they never took me up on it.  They have started getting off results on their assay too, so now they are working on repairing the plate reader.  I've offered the S.F. I was using to test fluorescence for their use, if they choose to.
The alternate plate reader I'm using is giving me consistent, expected results so I'm just going to continue with it  (it's more convenient to my lab too). 
Thanks,
Kelly

jackiemorris01
jackiemorris01's picture
Glad to hear things are

Glad to hear things are looking up, keep cheerful!
J