I'm trying to combine Analysis of Apoptosis and Cell Cycle Analysis using flow cytometry. I'm using mouse multipotent neural progenitor or stem-like cells (C17.2) and have access to a BD Canto II machine (3 lasers/ 8 colors). I'm very new to flow cytometry.
I've already tested the combination Annexin V and PI staining - Stain the live cells with Annexin V, fix in 70% ethanol and stain with PI. The Annexin V staining wasn't sufficient in our adherend cells and the needed fixation (we used 70% ethanol).
Do you have any suggestions on how to combine these Analysis (in terms of dyes or fixative...) or is it better to do two seperate staining?
Dear Tessa,
I hope the following link might be of some use to you Tessa. (specially Despina Smirlis answer)
https://www.researchgate.net/post/Can_apoptosis_be_detected_by_cell_cycle_studies
Hi Tessa,
How did you detach the adherent cells before attempting Annexin V staining? Many enzymatic methods degrade extracellular proteins, so one could assume that something like trypsin may have affected phosphatidylserine on the surface, thereby decreasing Annexin V binding. Perhaps switching to something like Corning CellStripper to de-adhere your cells could help. Otherwise, there are alternate ways to assess apoptosis that are amenable to flow cytometry, like dyes that bind to activated caspase 3/7.
@Preethi Pallavi M.C
Thank you for this link! I've already checked it. I don't want to detect Apoptosis with Cell Cycle Analysis. I would like to check for Apoptosis with a special assay but in combination with a Cell Cycle Analysis so that I can get the results for every single cell and use only the very healthy cells for Cell Cycle Analysis.
I would like to gate the cells in the following order. Maybe this makes my goals more clearly:
If it is not possible to combine Analysis of Apoptosis and Cell Cycle Analysis I would use separate Assays and just combine the Cell Cycle Analysis with a viability staining.
@Dominique Perez
Thank you for this explanation!
I trypsinized the cells to detach them. We already tried other substances fot detaching the cells but it wasn't sufficient because we could not get a good single cell solution with the other methods.
Maybe your suggestion to use a dye that binds to activated caspase 3/7 is better for my cell line. Than you very much!
@Andrea Maggioni
Thank you for this paper and the note!
I will definitely do more research on this in order to get an idea if this method would also work for adherent cells.
Kind regards,
Tessa
TUNEL assay if you do not specifically need Annexin-V : it plots Apoptosis against cell cycle.
Analysis of apoptosis by cytometry using TUNEL assay.
Zbigniew Darzynkiewicz, Dariusz Galkowski, and Hong Zhao
Hi Tessa,
ANX V/PI is vital assay, no fixation (Annexin V bids PS and after etoh fix you will lose most of the signal). PI is there do detect necrotic, secondary necrotic or late apoptotic cells, not for cell cycle. Nice modification of ANX/PI protocol here - it really works :)
https://www.nature.com/articles/nprot.2016.028
if you want o thave really complex protocol - check out our paper in Cytometry Part A
Article Multiparameter cytometric analysis of complex cellular respo...
good luck!
Karel
We have used EDTA for detachment and dissociation of neural progenitor stem cells from their matrix with very good success.
I takes a little bit of patience and can be done in Ca-free complete media to keep cells happy.
If you want to do live cell cycle analysis you could use some of the "Vybrant Dye Cycle TM" dyes that are said to be live cell permeable (I do not have experience with them in stem/progenitor cells).
You should be able to get a suitable pair sorted out.
You could also use some of the genetically encoded cell cycle sensors.
For a good overview article please see:
Article Classic Broken Cell and Newer Live Cell Methods for Cell Cyc...
Good Luck!
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