I have transfected 293 cell line with mouse CD47 and GFP (in same plasmid but gets cleaved apart later). I am trying to prove that the CD47 have been transported to the cell surface and functional. The only antibody I found that is specificly anti-mouse and doesn't cross with human have only 2 colours, FITC and PE. For PE I need to do compensation with GFP, and therefore another marker for 293 as a control.
I might be able to use another antibody, but I am worried that it would cross over with the human cells, and I rather use this one where it worked for my western blot.
1. Does the antibody come unconjugated? You could conjugate whatever fluor you'd like to it yourself. It isn't hard. In fact, it must come that way, since you used it for a western. Check out Invitrogen http://www.invitrogen.com/site/us/en/home/References/Molecular-Probes-The-Handbook/Antibodies-Avidins-Lectins-and-Related-Products/Zenon-Technology-Versatile-Reagents-for-Immunolabeling.html
You don't need a marker for HEK cells. It's a line. Therefore that's all you'll have in your dish. You simply need to separate GFP from not and then CD47+ from not. Unless I am missing something here. Feel free to elaborate if I am...
I suppose I just wanted something to do the compensation for the FACS on, so any surface protein on 293 cells will do, I sorta just used the word marker out of habit. I think I can label with APC, I hope it works since I have never done it before.
The presence of mouse CD47 will reduce mouse macrophages from engulfing these cells, this gene will later be packaged into adenovirus into another cell type though.
If you like, you can use beads (IgG-coated) for your comps. I use them all the time--particularly when I compare multiple tissues with very different autofluorescent properties and cellular contents. The main point of single-color controls is to have a negative and positive that are well-separated. It is best to comp on cells with similar properties, so that the matrix algebra that calculates values is correct across all colors. Thus, use unlabeled HEK cells to set your initial PMT voltages such that they fall between 10^0 and 10^1 and then use the beads as single color controls at these settings.
When you run the beads, you'll probably have to raise your forward scatter about 2-3-fold, and decrease side scatter to optimally "see" the beads.
Then, comp as you would with cells. I think you'll be impressed.
BD and Invitrogen both sell these beads.
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