FACS: DNA content and GFP in one go!!! - PI masks GFP??? (Jul/29/2005 )

Hi all,
I am doing FACS analysis of cells transfected with bicistronic GFP construct. I want to analyze DNA content of the cells(expressing GFP) to see cell cycle phases. First to gate GFP expressing cells and see DNA content. But when I stain the cells with PI and aquire it to see GFP, there are hardly any GFP expressing cells. And I can detect those, suspended in same buffer without PI(unstained). It seems PI masks GFP?????
Whether my protocol has to do something:

STAINING
1. Place 1x106 cells into each tube. (These are transfected with bicistronic GFP construct)
2. Spin down samples and remove supernatant as completely as possible without disturbing the pellet.
3. Add 1 ml of the hypotonic DNA staining buffer to the pellet and mix well.
4. Keep samples at 4°C protected from light for 30 min or for a maximum of 1 h before acquisition on the flow cytometer.

Hypotonic DNA staining buffer:
Sodium citrate 0.25g
Triton–x 100 0.75ml
Propidium iodide 0.025g
Ribonuclease A 0.005g
Distilled water 250 ml

Please, suggest me some ways or refer me such protocol to get rid of this problem. Thank you in advance.

What do you use for fixing your samples? Did u Check the solubility of GFP?

I have not checked the solubility of GFP. And I generally harvest cells, washed with PBS and add the buffer:

Hypotonic DNA staining buffer:
Sodium citrate 0.25g
Triton–x 100 0.75ml
Propidium iodide 0.025g
Ribonuclease A 0.005g
Distilled water 250 ml

After 1/2 hr, I aquire cells. There is no need of fixing the cells in this protocol. There is one protocol, where fixing is done with ethanol(final 70%). whether that will do? and what exactly is "solubility of GFP"?
Thanks in advance

I think it's because PI and GFP has the same excitation wavelength. With the addition of PI, it will compete for energy at the 488nm wavelength, so you'll have less GFP excitation so less GFP emission at the end.

I'm in the process of choosing a good dye for GFP emitting cells as well.

mklaw

There is no "competition" for excitation energy (you're using a high powered lazer to excite them, after all). As long as two fluorophores EMIT at different wavelengths and you're using an emission filter (the case with most flow cytometers), it's fine. GFP tends to leak out of cells over time, especially if it's not fused to anything. My guess is that your "hypotonic staining buffer" is actually lysing the cells, or making them more permeant, so the GFP leaches out (this is why you can detect GFP without staining). PI is also not normallly permeant into live cells (PI staining is often used as a marker for apoptosis), which convinces me that your buffer aids permeability of the cells.

You may want to try fixing the cells in paraformaldehyde (which will crosslink some of the GFP to stick inside the cell) prior to permeabilizing and staining with PI, or (probably the better option) treating with a cell-permeant nucleic acid dye like sytox green or hoechst 33242 in a isotonic buffer on live cells before you FACS them.