My questions are the following:
1) I first have my FACs profile, SSC-A vs FSC-A, I then have two plots that follow to remove doublets (FSC-W vs FSC-H, SSC-W vs SSC-H). I wanted to know if my P1 gate in the SSC-A vs FSC-A plot should encompass all of my cells, live and dead in order to make my Annexin V vs PI plot? Or should the cells in this plot only be live and not dead cells/debris?
2) For compensation, can beads be used for Annexin V? For PI, cells need to be used, but when compensating is there a specific population that needs to be selected in the SSC-A vs FSC-A?
1. The P1 gate should encompass all the cells excluding the debris at the bottom left corner. The entire protocol plus gating is provided in the ppt. You can refer the attached documents.
2. Compensation beads are polystyrene beads coated with antibodies that usually bind to the light chain of the antibody and are specific to the animal in which he fluorochrome conjugated antibodies are generated. So you cannot use it for Annexin V.
You can gate on a subset of cells in which you feel the antibody/stain will most likely be expressed at a higher frequency. While compensating, it is always best to use the brightest population on that fluorescent channel.
Pragun Muthya , wow, thank you so much, this is extremely helpful!
If your beads are labeled with antibody that conjugated same fluorophore with Annexin V, it's ok to compensate with beads. Alternatively, apoptosis positive control cells stained with annexin V alone can also be used to compensate.
For PI compensation, you can mix live and dead cells and then stain with PI. In FSC/SSC plot, gate cell population and in FL2 you will see to populations (PI+/PI-) to compensate. To kill cells, put them in 70 degree water bath for 30min.
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