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Cell cycle (phase) analysis by FACS analysis - PI staining to gate different phases of cell cycle (Jul/13/2005 )

I have stained cells (Huh7, Hepatocarcinoma) with PI and done FACS. Please suggest the ways to analyse the data so that I can do gating and get percentage of the aquired cells in each phase of the cell cycle (G0, G1, S, G2 and M).
Protocol is:
1. Place 1x106 cells into each tube.
2. Spin down samples and remove supernatant as completely as possible without disturbing the pellet.
3. Add 1 ml of the hypotonic DNA staining buffer to the pellet and mix well.
4. Keep samples at 4°C protected from light for 30 min or for a maximum of 1 h before acquisition on the flow cytometer.

Hypotonic staining buffer for DNA
Sodium citrate 0.25g
Triton–x 100 0.75ml
Propidium iodide 0.025g
Ribonuclease A 0.005g
Distilled water 250 ml

I have aquired 10000 cells. Histogram parameters are:
X axis: FL2
Y axis: Cell counts
X axis: FL3
Y axis: Cell counts
I am very new to this technique. And trying it first time. Please, guide me regarding the parameters (aquiring conditions) and data analysis.
Thank you.



I analyze my data like this (I use Hoechst, so you should adjust your detectors according to your instrument):

Dotplot1: FSC vs SSC (Gate1 viable cells)
Dotplot 2: FL1H vs FL4H to see excessive Hoechst dye which comes up at 520nm
Dotplot 3: FL4W vs FL4A to gate/separate single cells from doublettes (Gate 2 - single cells). This step is very important to later see the real ammount of G2-cells and not just 2 clumped cells.

You could have a look here to see what I mean:

then Histogram 1: G1-> G2 -> FL4-A which should in your case be FL2-A. It is important to use FL2-A instead of FL2-H. This may require pulse processing, depending on your instrumentation. In this histogram, you should see the typical picture of 2 peaks, the right one with double fluorescence intensity of the left one, separates by a plateau. To set the gates, you could use FlowJo or your intuition... If you want to make a proper s-phase analysis, using a second staining (BrdU or the new EdU from Invitrogen, which works really good at my hands) is necessary!
If possible, I would use Hoechst 33342 for this analysis if your instrument has a UV laser.

Good luck