I want to treat fibroblasts with 24h MG132 and then look a the amount of cell death. However, I want to correct for the amount of cells in total, because they also prolfirate in 24hour.
My supervisor suggested that after 24hour I stain my cells with HOECHTS and PI and then quantify with imageJ. The amount of dead cells can be calculated with:
x amount of HOECHTS positive cells/ x amount of PI positive cells.
However, HOECHTS also stains apoptotic cells. I do not know if this will interfere with my results. Do you think that after 24 hours most apoptotic cells are aleady dead anyway (or at least a part, normalizing the results)?
Or is this an unreliable method?
It should be reliable, especially if you are using one of the kits that are available. Just make sure to follow the times closely and wash at the end to prevent non-specific staining. The Hoeschst 33342 is cell permeable and binds to dsDNA in live cells regardless of membrane status. Propidium iodide (PI) will permanently bind to DNA in dead cells. Together, you should be able to tell normal form apoptotic and dead cells. Also, the blue (Hoeschst) and red (PI) are far apart so you shouldn't have much problem with overlap and you should be able to separate the stains into different color channels in ImageJ for quantification (https://youtu.be/i37VoiOQrnc and https://youtu.be/PqHFsmS1_JY).
You briefly mentioned proliferation - If you want to track proliferation over time then something like CellTrace (they come in various colors) passively diffuses into cells, is retained, and is inherited by daughter cells but is not transferred to adjacent cells.
Hope that helps.
- Melissa
Hi,
Hoechst staining should give you the number of live cells while PI will give you that of dead cells.
It shouldnt be a problem to use this method.
All the best.
Yes is quite reliable, I use this method often. Obviously HOE will stain live and dead, whereas PI only dead cells so you will have to count both and normalise. Then try and use an automated approach of counting cells with Image J so you can use the same parameters for multiple images and maintain consistency.
- Badges
- Science method
- Similar topics
- Biological Science
- Histology
- Staining