DAPI stains the nucleus but doesn't work well on apoptotic cells. Propidium Iodide (PI) works better if you are trying to analyze all cell cycle phases.
The peaks on the y-axis are the amount of cells, so the high peak at G0/1 is showing that there are lots of cells at this stage in the fell cycle. Along the x-axis is the DNA content so S phase is showing more DNA content than G0/G1, and G2 is showing more DNA content than S and G0/G1
For a fixing protocol where the cells are killed but preserved for imaging. This way you dont have to worry as much about decay of the cells before your analysis. Similar in principle to paraformaldehyde (PFA) treatment of tissue sections or microscope slides.
Presumably you dont have to though if you want to keep your cells alive. Prob put them in serum free media till you need to analyze, then prob swap out your media with PBS for less background signal.
This is also necessary for Propidium Iodide (PI) staining since it is membrane impermeable in live cells. Thermo has some nice non-toxic dyes that distribute throughout the cytoplasm and are membrane permeant to live cells. See CellTrace. They have three kinds, Violet, Yellow, and Deep Red.
Nice explanation. Thank you
This was great 👍🏻
Awesome 👌
Thank you sir very helpful
really helped in my campbell biology exercise
thanks so much
thankyou so much
Can you please tell me how much PI and Rnase should be there? Should we add sodium citrate with that?
Thanks
Hi I want to measure cell counting of sulfate reducing bacteria. Can you share any suitable protocol ?
Thank you so much. Would Dapi staining work the same?
DAPI stains the nucleus but doesn't work well on apoptotic cells. Propidium Iodide (PI) works better if you are trying to analyze all cell cycle phases.
but just to be clear you are still able to use DAPI but that phase might be a little inaccurate
How many cells per well if we use a 96 plate? 72h treatment... I know it varies, but my cells grow a lot, I wonder what you would suggest haha
How can we count the cell?? In cell cycle
Why doesn’t S phase have a higher peak than G0/G1? Cells synthesisze new DNA in S phase so there much be more DNA that PI binds to.
The peaks on the y-axis are the amount of cells, so the high peak at G0/1 is showing that there are lots of cells at this stage in the fell cycle. Along the x-axis is the DNA content so S phase is showing more DNA content than G0/G1, and G2 is showing more DNA content than S and G0/G1
@ 0:57 I swear I thought that was my phone😂
You resuspended in 2 ml ETOH?
For a fixing protocol where the cells are killed but preserved for imaging. This way you dont have to worry as much about decay of the cells before your analysis. Similar in principle to paraformaldehyde (PFA) treatment of tissue sections or microscope slides.
Presumably you dont have to though if you want to keep your cells alive. Prob put them in serum free media till you need to analyze, then prob swap out your media with PBS for less background signal.
This is also necessary for Propidium Iodide (PI) staining since it is membrane impermeable in live cells. Thermo has some nice non-toxic dyes that distribute throughout the cytoplasm and are membrane permeant to live cells. See CellTrace. They have three kinds, Violet, Yellow, and Deep Red.
How many cells is required for an analysis?
Several thousand at least for Flow Cytometry.
10k and it will take a LONG time with this little cells
Why are we degrading RNA during cell cycle analysis?