Need advice on FACS for cell cycle analysis - (Jun/16/2005 )

Hi there,

I am going to do FACS on cultured cells to determine cell cycle related parameter (no specific marker, just DNA content). I have never done such experiment and this experiment is critical for my whole project and I don't have much time for a preliminary experiment. I am going to follow protocol found here: http://www.flemingtonlab.com/Protocols/Pre...sforFACS-PI.pdf

After I prepare my cells I will give them to our core facility to do the measurement.

I will appreciate any advice, suggestion, precautions from you before I preceed.

Thank you!

[quote=green,Jun 17 2005, 12:47 AM]
Hi there,

I am going to do FACS on cultured cells to determine cell cycle related parameter (no specific marker, just DNA content).



dear green!

i don't really understand which cell cycle parameters you want to determine. i do FACS cell cycle analysis to estimate how many cells undergo different cell cycle phases. also, i performe different stainings to estimate apoptosis.


here is protocol for cell cycle phases:


Cell cycle staining

Vindel’s dye: 0,121 g Tris base
5,84 mg NaCl
1 mg RNA-ze (700 U)
5 mg PI
Novidet P-40 / Triton 5 µg pH→8,0
dH2O ad 100 ml

PI (propidium iodide) is a dye that is intercalated in the DNA molecule. Normally, it penetrates through the cell and nucleus membranes only when the cell is dead, therefore it is used to label dead cells. It is also used to estimate cell cycle, but in that case, cell and nucleus membranes should be permeabilized with some sort of detergent, which is usually already mixed with the stain.

Staining:
1. harvest 0,5-1 x 10E6 cells / mL
2. wash them in 1X PBS twice
3. discard the supernatant
4. add 1 mL of Vindel’s dye to 1 x 10E6 cells and using a pipette mix the suspension very, very carefully, otherwise it will foam!!
5. incubation 40 min, +4° C, in the dark
6. pellet the cells on 150-180 G, 6 min, but do not discard the supernatant
7. using a pipette remove about 400 µL of supernatant (carefully, in order not to remove the cells, because the pellet is not firm)
8. flow cytometry, 300 cells per second max.


Stock of Vindel's dye (i not sure about the spelling) should be used up within 30 days!

Number 8 is very important: 300 cells per second is really *the maximum*!


i don't know will this be helpfull. anyway, i can send you apoptosis assay also!

hello,
I agree with the earlier reply, though both vindelov's method and ethanol method will allow you to determine DNA content, ethanol may probably give you higher amount of debri.
you may probably need to standardize thr treatments for various phases if you are looking for cell cycle phases

good luck

Hi
I am also interested in using prpidium iodide staining to see cell cycle phases.
in the Vindel's recipe I don't understand some things:

5ug NP40/Triton? but they are liquid!

in staining step 7, says remove 400ul! Do you mean discard? and should the cells be resuspended again in the 600ul left to be acquired?
Thanks