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).
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.
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: http://www.ucl.ac.uk/wibr/services/docs/cellcyc.pdf
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.