I am doing cell cycle analysis using Propidium iodide dye in flow cytometry. My samples are
1) cells
2)cells+ silica
3) cells+ drug
4) Cells+ silica-drug
The cells treated for 24 hrs with the drug and silica-drug to the cells. The dead cells start floating after 24 hrs treatment. My doubt is that if I throw the media from the flask, dead cells also gone with the media. Whether I need to take the floating cells (dead) for the cell cycle analysis? Please note: my cells are adherent cells.
You should not discard the supernatant. Add the supernatant containing dead cells to the suspension of cells that were released by trypsin. Then you must centrifuge and resuspend the pellet with the buffer containing PI.
You should not discard the supernatant. Add the supernatant containing dead cells to the suspension of cells that were released by trypsin. Then you must centrifuge and resuspend the pellet with the buffer containing PI.
Hi Pra, you can detect cell viability and cell cycle, respectively. According to your description, drug treatment led to cell death. You can evaluate cell viability using MTT. Next, you detect cell cycle distribution using the live cells. If you want to combine them together, you can follow Vitor's suggestions.
Dear all and Han Zhang,
Thanks everyone for your answers. I want to do only cell cycle analysis using flow cytometry. My cells are adherent cells. For cell cycle analysis only, whether I need to collect the dead cells floating in flask or not?
otherwise I can discard the media containing dead cells in the flask, then I can collect only live cells for analysis?
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