On 29/08/2007, at 11:50 PM, Rosemary Clarke wrote: > Dear All, > > Now this is going to sound like a very stupid question, so > apologies in advance. > > Is it possible to detect mCherry fluorescence on a FACS Calibur, > using 488nm excitation and detecting fluorescence at 585nm? I have > always thought not as (in theory at least) mCherry is not excited > at 488nm. However, I have just had a user come to me who thinks he > may be getting a good signal (two log shift from untransfected > cells) with what they believe to be mCherry. However, there could > have been a mix up and it may be dsRed (don't ask!), which would > obviously be detectable. They are in the process of checking and > sequencing the constructs but I am now paranoid that I have got it > wrong and mCherry CAN be detected. Has any one out there in flow- > land managed to get a signal from mCherry on a Calibur using 488nm > excitation? > > Thanks in advance > Rosie > Hi Rosie In testing mCherry on our VantageDiva we can see some signal from the 488 (@200mW)... but it is poor, especially compared to excitation by the dye laser (or to GFP). Two logs on the Calibur seems unlikely, particularly as I think the dectection filter in FL2 on Calibur is also sub-optimal. Note that in attached plot we had to turn the O630_6 voltage way down (about 300V I think rather than our normal 600), ie this was a very bright sample... Regards, Adrian Received on Fri Aug 31 16:18:00 2007
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