thanks for suggestions about Annexin-v assay

Dear flowers,
  I got a lot of response about Annexin-V assay, the following are parts of them. 
  Thanks a lot for so many good ideas.
  Meixia
   I¡¯m assuming you are running your sample on a FACScan or FACSCalibur. With FL1 vs FL3
you may need digital acquisition to compensate. With PI you will get a signal from FL2
and FL3 due to it¡¯s spectral emission. I¡¯ve always used FL1 vs FL2 which lets me be in
control of the compensation setup. Make sure you have all your single controls with PI
and Annexin ¨CFITC. With FL1 vs FL3 you may not have control over the compensation
depending on the equipment you are using¡­
  Impossible to tell which is "better" without knowing what instrument you're using, and
what the emission filters are.
  However, compensation should not worry you.  Run unstained cells, and set your PMT
voltages so that your cell population falls in the first log decade (or partly against
the axis when looking at the red channels).  Measure the MEDIAN value of the population. 
Then run the SINGLE COLOR control (i.e. FITC-Annexin only, or PI only) on a FITC vs PI
bivariate.  Adjust compensation such that the MEDIAN value of the positive (control)
population matches the MEDIAN value of the negative population.  DO NOT CHANGE YOUR
VOLTAGES DURING THIS PROCESS, or your compensation values become wrong.  Repeat this
process for the other (PI) positive population.
    In general, using FL3 and FL1 is better than using FL2 and FL1, as the spreadover
from FL1 to FL2 is quite significant. 
  However, you still need to do the compensation even you use FL1 vs. FL3. Setting
compensation is the basic of flow cytometry, and compensation setting is also a function
of voltages. When you change your voltage setting, you also need to change compensation.
  Ordinarily FITC is in the FL1 channel whereas PI is in the FL3. Your requirement for
compensations of FL1 v FL3 depends on the instrument that you are using. The BD
FACSCalibur does not require this compensation, whereas the Coulter FC500 does
  FL3 has less spillover from FL1 than does FL2, so sometimes is better.
Both may require spillover correction.

   am doing similar experiments now and using more colors. In my 
opinion, PI is better for FL3 (which machine you are using?).  
Compensation is necessary no matter for which combination. If you are 
studying cell line, you don't need a surface marker, but for fresh 
cells, it's better to have one.
Another important thing is whether your FACS machine could gate out 
doublets. It always interfere with analysis.



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