FITC/APC pair vs FITC/Cy5 pair, which is better? - (Dec/25/2008 )
Hello there,
I am new to Flow cytometry and my boss has asked me to do a dual-channel FACS. I have made a good progress with FITC(green), now I'm considering staining my sample(biotin labeled DNA) with a second dye.
Currently there are 2 candidates in my mind, APC and Cy5, both are streptavidin conjugates. Their spectrums are similar (Ab/Em=650/660 for APC and 650/670 for Cy5), and we have a Flow cytometry with 488nm/633nm lasers available.
I know that the quantum yield of APC is higher(0.68) than that of Cy5(0.28), does that mean I would get a stronger signal from 675nm channel using APC? I also heard Cy5 could only be excited to about 63% of max with a 633nm laser, but the molecular weight of Cy5 is much smaller than that of APC and it's also much stable and suffers less from autofluorescene.
Can anyone tell me which will work out better? I simply couldn't persuade my boss to buy me both!!
THANKS A TON!!
Hi,
This link is about fluorescence microscopy, but i think you might find it helpful anyway.
http://www.olympusfluoview.com/java/dualprobes/index.html
you need Java to use the tutorial.
Good Luck!
This link is about fluorescence microscopy, but i think you might find it helpful anyway.
http://www.olympusfluoview.com/java/dualprobes/index.html
you need Java to use the tutorial.
Good Luck!
That tutorial is just AMAZING! Thanks again Gat, you've been so helpful.
The FITC - APC pair is more commonly used in flow cytometry. FITC - Cy5 co-staining was used frequently in fluorescent microscopy however the advent of the Alexa-Fluor dyes is much more prevalent in fluoresent microscropy now.
I would highly suggest using FITC and APC in your experiment. Your boss may want to move beyond two colors and there are more reagents available with APC hooked up rather than Cy5. Furthermore, if you do go to multicolor flow and begin using some of the tandem dyes (i.e. PE-Cy5) you will not be able to use Cy5, but would be able to continue using APC.
Happy flowing.
There are numerous online flow groups that would also be able to help you with flow cytometry - search for or link to New England Users Group http://www.bostoncytometry.org/NEChomepage.html and the Purdue Cytometry mailling list http://www.cyto.purdue.edu/hmarchiv/index.htm
I would highly suggest using FITC and APC in your experiment. Your boss may want to move beyond two colors and there are more reagents available with APC hooked up rather than Cy5. Furthermore, if you do go to multicolor flow and begin using some of the tandem dyes (i.e. PE-Cy5) you will not be able to use Cy5, but would be able to continue using APC.
Good point. However, there's one more thing that I'd like to hear your expert opinion. If APC is to be used, 2 large protein groups (mAb&FITC/Streptavidin&APC) will come very close to each other (the distance between them will be less than 7nm). I'm afraid possible effect of steric hindrance may plague affinity binding of the target, which is the main interest of my research.
Sorry if I've gone too far, perhaps I was just over-thinking it.