Protocol Online logo
Top : New Forum Archives (2009-): : Flow Cytometry

Fluorochromes for FC and microscopy? - (Dec/03/2013 )

Hey all,


I'm trying to find a fluorochrome in the red channel that is suitable for both FACS and microscopy. Basically, I'm sorting cells using the Influx and then want to visually confirm they are what I think they are using a microscope afterward. Alexa 488 works well for the green channel but I'm having a bit of trouble finding something suitable for the red channel. I'm using an antibody clone that works in both FACS and IHC so I think it's only the fluorochrome that will be an issue. I know not all are suitable. For example, PE is great for FACS but photobleaches within seconds on the microscope. eFluor 650 seems to work in both but the data sheet from the antibodies explicitly say that they are not suitable for both techniques. Is this legitimate? Are there really differences in the antibody preparation if they are designed for FC vs IHC, assuming same clone, same fluorochrome, same company?


Thanks for any insights!




Have you tried Alexa594 or alexa633?


I know there are a lot of fluorochromes that would hypothetically work, including Alexa594 or Alexa633. I guess my real question is whether an antibody with an alexa fluorochrome (or other stable fluorochrome) would work both in flow cytometry and under the microscope despite vendor warnings not to. (I only have Alexa488 to test in the lab and I know it works but thought I would check on here so that I don't end up buying antibodies in other colours for nothing).


I work a lot with AF-647 on the fl-microscope.  It is a very popular dye conjugated to antibodies, so it should not be a problem to just buy it on different antibody clones and test them on flow and f-microscope.  However, you may know that dyes in that range are hardly detectable by naked eye.  You most likely need a camera on your fl-mic to visualize the sample...

By the way since you mentioned the PE, if you can find a conjugate with AF-555, I think it'll make your life much easier, but for whatever reason this dye does not appear much as an antibody conjugate...I use streptavidin conjugated AF-555 against biotin and it works great...but I imagine this is not what you need..


I used Alexa-647 a lot in FACS and there it worked fine for me. I don't know why it shouldn't be detectable with a microscope (never tried it though), unless your target is expressed in an extremely low amount.

And I have always heard that the Alexa dyes have a higher photostability than the classical ones, but again thats something I never tried myself.


I thought the reason the alexafluor ones weren't used for FACS is that often the lasers weren't of the right wavelength to excite the fluorophores.   However, with most modern FACS machines this isn't a problem, and while I don't do FACS at the moment, I know of lots of people using Alexafluors with FACS.