[Cytometry] PerCP exitation by Alexa 488.
roederer at drmr.com
Thu Jun 17 08:21:08 EDT 2010
Nothing to do with saturation...
A 20 mW laser will have nearly 10^12 photons per 10 microseconds (cell's illumination time). If you have 100,000 Alexa molecules, these will generate perhaps 10^7 emission photons during the same 10 microseconds. So even if all of those were available for absorption by PerCP (and they wouldn't be), you are many orders of magnitude below what the laser has. (This is all back-of-the-envelope, forgive my estimations).
But... still... why would this be a "false positive"? If the PerCP is present in the cell, then it's a "true positive", right? And if there's no PerCP on that cell, then you have nothing to worry about. So all my (mistake-laden) calculations are, in fact, irrelevant...
On Jun 17, 2010, at 3:48 AM, Dmitri Lubenets wrote:
> Dear flowers,
> I have a question about Alexa 488 and PerCP fluorochromes. Looking to their
> exitation spectra the one can see that A488 emission could exitate PerCP
> protein. I wonder if the system where single cell is stained with A488 and
> PerCP conjugated antibodies (ran on FACS Aria I equipped with 488 laser)
> will give false positives due to exitation of PerCP by A488 emission or this
> is not relevant because fluorochromes will be saturated by 488 laser anyway.
> Thank you in advance.
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