EMBL FLOW CYTOMETRY CORE FACILITY
Cell Cycle on the FACScan Flow Cytometer
Cytometer Self Help Guide:
By ANDY RIDDELL
Revision No. 1.0.5
This document is intended to familiarise the reader with the setup procedure for cell cycle in the FACScan
This Guide will introduces the reader to:
Setup the software correctly,
Adjust the Hardware to reduce error in data acquisition.
NOTE: Please read the guide: Acquiring data from the Becton Dickinson FACScan Flow Cytometer.
The FACStation software has MACRO setup to simplify, unify and speed up sample acquisition.
F7 will display a simple one dye DNA setup such as PI or 7AAD.
F8 will display a DNA dye and a fluorochrome such as PI and GFP. Please read: Cell Cycle Plus colour On
the FACScan Flow Cytometer Cytometer Self Help Guide.
SETUP THE SOFTWARE: F7
On the FACStation, press the key marked F7 on the keyboard. A one stain set will display. Allow it to finish. If
you see any error messages, please read the guide Acquiring data from the Becton Dickinson FACScan Flow
Pressing F7 on the FACStaion keyboard will display this layout.
Follow the instructions on page 5 of the guide Acquiring data from the Becton Dickinson FACScan Flow
Put your sample on the SIP and press “Acquire” on the “Acquisition Control” –make sure that the “Setup” box
is ticked”. The “Acquire” button will change to “Pause”.
Acquisition Control with Setup checked and Acquire pressed.
Adjusting the scattering:
On the Computer look at the FSC vs. SSC dot plot. You should scan the axis of the plot to see if you can see
some dots or a line.
Figure shows FSC vs. SSC dot plot when the sample is placed on the SIP.
From the picture above, you can see the cells on the origin of the SSC axis but just away from the origin of the
FSC axis. Look all around, if you have very big cells they can appear as a dot in the bottom right corner. Just
adjust the FSC voltage in the Detector/Amps pallet to bring the cells on scale.
Figure shows Detector and Amps pallet with Voltage drop down menu.
On the Detector Pallet in the row labelled P1 select the drop down menu (it initially reads E00). For cells with
bigger or smaller FSC scattering you can select here the appropriate setting.
Note: E-1 will give 1/10th. the amplification of E00. This is for cells with large FSC scatter. E01 will give 10
times the amplification of E00. This if for small cells with small FSC scatter. E2 will give 100 times
amplification of E00 and E3 will give 1000 times the amplification of E00.
We want the cells to be in the middle of the FSC plot (later we will draw around the population). Still looking
at the Detectors/Amps pallet Adjust the Amp Gain arrows until the cells are between channel 200 and channel
Figure shows Amp Gain at 4.26 for FSC parameter.
Note: If you click the centre of the Up Down arrows you will get a slider.
Figure shows cells between the channels 200-800.
Now we alter the SSC parameter P2 in the Detector/Amps pallet.
Adjust the Voltage of the parameter P2 to place the cells between channels 200-800 on the SSC axis of the FSC
vs. SSC dot plot.
Figure shows SSC adjustment.
NOTE: Clicking on the middle of the Up/Down arrow box will give you a slider.
Figure shows FSC vs. SSC plot containing cells between channels 200 and 800 for both FSC and SSC.
On the Acquisition pallet click on “Pause”, then click on “Abort”.
Looking at the Detector/Amps pallet tick the DDM Box
Figure shows the DDM of the Detector/Amps pallet ticked.
Now the DDM parameters are selectable.
Figure Shows FL2-A and FL2-W Adjustments accessible.
The Dot plot to the right of FSC vs. SSC now shows FL2-A vs. FL2-W.
On the Acquire pallet click on the Acquire button and start acquisition the setup box is checked.
Drawing a Region:
On the tool pallet select the polygon tool.
In the figure the black arrow points to the polygon tool.
The cursor will change to a ‘+’ sign. On the FSC vs. SSC dot plot start at the top, click once to anchor the line
on to the plot and by clicking at appropriate points draw around the healthy looking population.
Figure shows a region drawn around the healthy population.
Look at FL2-W vs FL2-A plot.
Figure shows dot pilled up on the axis.
On the Detector/Amps pallet make an adjustment to the P4 FL2 voltage.
Figure shows adjustment to FL2 detector.
Position the dots thus:
Figure shows G0/G1 population at channel 200 on the FL2-A axis.
Now adjust the FL2-W parameter (P7) in the Detector/Amps pallet so that the G0/G1 population is at channel
200 in the FL2-W axis.
Figure shows P7 adjusted.
The FL2-W vs. FL2-A plot now should look like this.
Figure shows FL2-W and FL2-A Go/G1 population at channel 200.
From the Tools pallet select the polygon again and draw around the singlet population.
Figure shows Singlet population drawn around.
Gating on the Histogram:
From the Gates menu choose Gate List.
Figure showing the Gate List Dialog box
Ensure the entry in G8 definition is as above. Close the Gate List dialog box.
NOTE: The Definition is case insensitive. Do not use a ‘+’symbol as this is an ‘OR’ instruction. Type the word
Look at the Histogram labelled FL2-A and select it by clicking on it.
Figure shows G0.G1 peak around channel 200.
In the Plots menu select Format Histogram. Then on the Gate drop down menu in the Histogram Plot dialog
select G8 = R1 and r2.
Figure shows the Histogram Plot format dialog.
Close the Histogram Plots Dialog
Now looking again at the FL2-A histogram, the G0/G1 population should be centred over channel 200. To do
this we need to make another correction.
On the detector/Amps pallet we will make and adjustment in P6 FL2-A.
Figure shows the adjustment in FL2-A in the Detector/Amps Pallet.
Adjust the G0/G1 population until the G0/G1 population is centred on channel 200.
Figure shows G0.G1 population at channel 200.
We are now ready to acquire data For the F7 single DNA stain.