USE OF FLOWJO TO ANALYSE CELL CYCLE DATA
FlowJo software has been loaded onto a number of computers in the BAIR computer
facility. If you require this software for your own computer ; please request the CD
from BAIR personnel. It should be noted that the software is dongle registered and
can only run with the dongle; this is also available in the BAIR but must be returned
after use (we only have one copy). Failure to do so will result in the right to use it on
your own computer being removed.
1. Double click the FlowJo icon on the desktop( FlowJo 7.2.2)
FlowJo
icon
2.Workspace window appears.
3.Click on add sample button.
Add
sample
4. Select folder containing files to be added, and click open
Click
‘open’
5 The files will be automatically loaded onto the workspace.
6. Double click your first data sample and the following plot appears
Double
click
FSC/SSC
plot
appears as
default
7. Draw a region around your whole cells using the indicated drawing tool
Use
polygon
drawing
tool
8. This creates a subset , which is added to your data list.
Subset of
your data
9. Double click on the subset and the following plot appears. From this plot
change the axes to FL2A and FL2W.
Change the
axes on this
plot to
FL2A and
FL2W
10. The resulting plot can then be gated to eliminate cells that have aggregated
using the polygon tool.
Draw region
around cells
of interest
11. The subset created is added to your data list. Right click on this subset to bring
up a menu. From this menu choose ‘add cell cycle’
Right click
on subset
and choose
‘add cell
cycle’
12. The cell cycle profile then appears.
RMS
Cell cycle
profile stats
Models
Constraints
The cell cycle histogram contains statistics giving percentages for G1, S and G2
phases of the cycle. There are 2 algorithms to choose from; WATSON or DEANJETT-FOX. Both of these models fit Gaussian curves to G1 and G2 ; however the
Dean-Jett-Fox model fits S phase to a mathematical model but Watson does not.
The fit of the model can be assessed by comparing the model to the DNA
histogram and by a low RMS (root mean squared) score; the RMS is found with
the statistics. If the model does not fit either change models or adjust the
constraints to obtain a better RMS.
13. The subsets and cell cycle from first data sample can now be applied to all the
samples in the data set. Highlight the subsets and cell cycle and drag them into
the folder containing all the samples.( i.e blue writing)
Highlight
subsets and
cell cycle
14. All samples will then contain the gates you have applied. These will appear as
blue.
Gates
applied to
other
samples
Arrow
buttons for
clicking
through
samples
15. On the cell cycle histogram you can cycle through each data sample by
clicking on the arrow buttons.
NOTE: when copying the gates into your other samples please check that these
gates do encompass the cells you are interested in. Click on the subset you are
interested in and go through each sample using the arrows.