Re: Compensation for Annexin-V/PI assay

Meixia:
Hi!
PI will be detected inFL2 and FL3 strongly, so use the one where you see it best. sometimes PI is so strong that it comes off scale, so use the other channel in that case. In general, I prefer to use FL3 because compensation is easier because it is smaller. FL1 spills into FL2 usually ~15-20% depending on your settings, but it spills only slightly into FL3. You have to compensate FL1 vs FL3, that you'll see from your controls - you should run an Annexin FITC single stain and a PI single stain, as well as an unstained. Use them to determine your compensation. If you are only analyzing subpopulation percentages it is tempting to use compensation "by eye", but that is a bad habit to learn. If you work with dead and dying cells, it is surprising the kind of strange fluorescence shifts you can get sometimes. If you did not compensate with the proper controls, you can get into a lot of uncertainties that can ruin your results.

Good luck,

Uriel.
Uriel Trahtemberg, M.Sc.
MD/PhD student
The Laboratory for Cellular and Molecular Immunology
The Hebrew University - Hadassah Medical Organization
Jerusalem - ISRAEL
  ----- Original Message ----- 
  From: Meixia Zhang 
  To: Cytometry Mailing List 
  Sent: Thursday, November 16, 2006 8:21 AM
  Subject: Compensation for Annexin-V/PI assay


  Dear Flowers,
   
  I am planning to run FITC-Annexin-V/PI staining. Which one is better FL1vs. FL2, or FL1 vs. FL3? Also I have a question about compensation. If we use FL1 vs. FL2, I should run compensation. Is it necessary to do compensation if I use FL1 vs. FL3?
   
  I appreciate your comments
   
  Meixia