Hi All,
Can anyone tell me how much Propidium iodide, RNAase A and Triton-X 100, we use to make cell cycle solution? Thanks.
Here is a rough protocol for performing the assay using adherent cell culture.
1. Harvest cell with Tyrodes solution (or EDTA) followed by 0.25% Trypsin.
2. Neutralize with medium containing FBS, centrifuge.
3. Wash 1X with PBS, centrifuge.
4. Add 750 µl 0.1% Sodium Citrate + 0.1% Triton X-100 to the cells at a concentration of 2X10 6 cells/ml
a. 0.1% Sodium Citrate + 0.1% Triton X-100 = 0.1 gm sodium citrate (mw 294.1) + 100 µl Triton X-100 in 100 ml ddH 2 O; warm to dissolve
5. Add 250 µl Ribonuclease A (final concentration=1mg/ml) to the bare nuclei in the Sodium Citrate/Triton X-100 solution.
a. Rnase A stock = 4 mg/ml; dilute 1:4.
6. Incubate RNase A solution for 30 minutes at 37°C.
7. Add 10 µg/ml propidium iodide
a. stock propidium iodide = 120 µg/ml, therefore dilute the stock 1:12 by adding 90 µl to 1 ml bare nuclei solution
8. Cover in foil and keep at 4°C until analysis.
Hi Laychiluh,
Anne's protocol looks good, and I'm sure it works. Just to provide an alternative, this is what I use (which is a little shorter than Anne's):
1) Lift your cells with Trypsin/EDTA, quench trypsin with an equal volume of media containing FBS
2) Spin down the cells at 500xg, 5' and wash twice with ice-cold PBS
3) Resuspend your cell pellet in 0.6mL ice-cold PBS then add 1.4mL ice-cold Ethanol (100%) dropwise on a vortex (put your tube on a vortex and, while vortexing, add the ethanol, drop by drop, over about 30 seconds). You can store the cells for up to a couple of months at -20 degrees in this ethanol
4) To use, spin down the cells (1000xg, 5') and wash twice in ice-cold PBS
5) Resuspend the pellet in 200uL of PI Buffer (see below), leave to incubate on ice for 30 minutes (protected from light) then analyse by FACS.
PI Buffer (10mL)
10uL of Triton X-100
200uL of 1mg/mL propidium iodide solution
2mg of DNAse-free RNAse A
Make up to 10mL with 1x PBS.
Hope that helps! Let me know if you have any questions.
Cheers,
Dan
Thanks a lot Anne and Dan for the tips, its really helpful.