I am using an LSRII w/ FACS DIVA software, 405/20bp for Indo-violet and 525/50 505lp for indo-blue.
What should I have the scaling factor set to for the Ratio of Blue/Violet fluorescence?
Is there an optimal approach to setting the PMTs for both channels?
If you are working with cardiac myocytes, you may try excitation 340nm. Then measure emission at about 400nm and 500nm respectively (R400:500). Play with your system and see what works best. You may need to use some ND filters in front of your excitation light source. Find out what gives you the best signal to noise ratio. You do not want to photo-damage your cells too quickly, but you do want a decent (Bright enough) excitation light. For guidance you should be able to experiment for about 2 h with the same cell with minimum bleaching. You will also need to estimate your cells' autoflourescence or background signal. Again, if you are working with isolated cardiac myocytes I can refer you to my paper: Diaz et al, 1997. J Physiology, 501(1): 3-16, for further details, particularly in case you may want to calibrate calcium. I hope this helps.
Best wishes. Mary
Thank you Mary, that was helpful. In regards to setting the PMTs, I found this on the purdue cytometry list, by Ron Rabin: " I have done a lot of calcium flux on the LSR2. depending
on the cell type, there are always some cells with high concentration
in the background. The more activated they are, the broader your
baseline. Resting human T cells give a very narrow baseline.
Activated T cells kind of broad. monocytes narrow, macrophages broad.
One suggestion: since the digital platform collects all the data as
linear whether your scale is log or linear, collect it on the log
scale, making sure that blue is 1-2 decades above unloaded cells
(which will be on scale in log) and violet is at least 1/2 log above
unloaded cells. You end up with a wider range of collection and you
are not fussing with PMTs as much trying to figure out the best
baseline."
ron
Hi! I am sorry to be asking such a basic question but how do I make 2 emission measurements with a single excitation? I am using clampex to record and I think I have the same PMT settings. I never had experience using Indo-1 before so I would appreciate any help on this. Thanks!
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