Gating of annexin V PI assay - (Jul/28/2010 )
Hi all,
For annexin V PI assay, which regions should I gate for the FSC VS SSC? Only viable cells, or viable cells together apoptotic cells?? Thanks
Samantha
I guess it depends what you're looking for/at!
Healthy, apoptotic and necrotic cells should all sort out in to different populations when you compare the fluorescence (as long as the flurophore attached to your Annexin V antibody fluoresces at a different wavelength to the PI).
Apoptotic cells will/should begin to expose phosphatidyl serine molecules which the Annexin V binds to.
Necrotic cells will take up the PI.
Healthy cells shouldn't be affected by either.
Could you explain a bit more that? Actually, I would like to see if my gene would sensitize drug response. THanks.
Samantha
When you treat your cells with Annexin V and PI and then run them on the flow cytometer, the cells will fluoresce at different wavelengths according to what they have taken up/bound to the cell surface.
PI fluoresces at 562-588nm when excited with 488nm wavelength light. Thus if you have necrotic/dead cells, they will take up PI and will fluoresce accordingly.
Annexin V is marketed with several different flurophores. I use an Annexin V that is conjugated with FITC (Fluorescein Isothiocyanate). This may also be excited with a wavelength of 488nm light and has an emission peak at 521nm.
The detector on your flow cytometer should be able to differentiate between these two emissions and so will be able to plot this data. For example, if you set your y-axis as fluoresence around PI (562-588nm) and the x-axis as fluoresence around FITC (521nm) you will find healthy cells have a low fluoresence and will be found at the bottom left quadrant. Apoptotic cells will have a higher fluoresence on the x-axis (events appear towards the right) and necrotic cells will have a higher fluoresence on the y-axis (event appear towards the top).
It is always wise to also run a NIL sample in the same way you prepare your positive sample to check on the auto-fluoresence of your cells so that you can compare the two and distinguish truly apoptotic/necrotic cells.
I will try and find an example from my date to show you this. Also I assume you included some Calcium in your Annexin V prep? Annexin V needs Calcium to bind at all. I use 5mM CaCl2 in 1xPBS.
Here's the example...
Thanks for your reply. Do you mean I should gate both apoptotic and viable cells for FSC VS SSC??
Samantha
It depends on what you're looking at!
If you're only interested in 'healthy' cells then gate on this population on the fluoresence plot (in my example, bottom-left) and have the FSC vs SSC plot only show these events.
On the contrary, you could gate specific populations on your FSC vs SSC and see how these populations fluoresce in terms of binding Annexin V and/or PI.
Yes, you should include some of the presumed-apoptotic region (towards lower-left of the FSC-SSC plot); if using adherent cells, you should also remember to include floating cells in your analysis. You'll have to decide how small to go, but this can be autamatically cut off by your threshold setting (ex: FSC @ 55) to exclude the most-fragmented debris. Ideally, your assay should be performed at a time point wherein the cells are still fairly intact... wait too many days, and they will fall apart. The plot submitted by Piersgb is, in my opinion, an example of a poorly run assay. When both labels are used (Annexin V- FITC plus PI), there should be almost no PI-only population.
Healthy cells: negative for both; lower-left quadrant.
Early apoptotic: lower-right quadrant. PS exposure, and thus Annexin V- FITC binding, increases as apoptosis advances. The cells will continue to try to exclude PI as long as they are alive. Position on the PI axis may sart to curve up as annexin signal increases, but there will probably be about a log-order of difference between these the centroids of the popoulations of early apoptotic cells and the truly dead ones.
Dead (fully apoptotic, or possibly necrotic, depending on treatment): positive for both, upper right quadrant.
Consult the Purdue flow listserve for a better summary:
http://www.cyto.purdue.edu/archive/flowcyt/research/cytotech/apopto/data/chap16.htm
One addititional tip: when your methods tell you to count cells and use a certain number for the staining, it really is meaningful to follow the instructions. From my experience, adding a vast excess of cells (eg, millions instead of 100k) can result in sub-saturating concentrations and will skew your data with understaining of PS leaflets.
-Eric