Beckman Coulter Cytomics FC 500 User Manual

USER GUIDE TO BECKMAN COULTER FC500
Create a New Protocol
Purpose: This procedure describes how to define the parameters and create rough
analysis plots for a new experimental set-up. Some protocol templates with 1, 2, 3, 4, or
5 fluorescent parameters exist in the CXP/Users/FLOW/AcquisitionProtocols folder,
which can be modified instead of creating a new protocol from scratch.
1. Open CXP Software and log in
2. Click File, then New, then New Protocol
3. Click Cytometer then Cytometer Controls
4. In the Acq. Setup tab, adjust the Discriminator, as needed (most mammalian cells
= FS 60-100; bacteria = FS 2-5). Also adjust Max Events, Duration, Red Laser
Shutter, Live Gate, and Dots as needed.
5. Click Parameters. Select the desired parameters by checking or unchecking log
or lin. Order of parameters can be altered in the Selected Signals Box.
6. Click OK. The program will prompt you to rename the protocol. Type in the
desired name, then click OK. Leave the Cytometer Controls window open for
future use.
7. Name the Axes: Click File then Edit FCS Header Attributes. Click on FL1 and
type in the fluorochrome for it Name. Continue with FL2, etc. Click OK when
finished with naming.
8. Create Plots: Dot plots and/or histograms with appropriate axes by selecting the
desired plot icon from the toolbar or by clicking Plots and then choosing the
desired plot. Select the appropriate parameter for each axis.
9. To easily arrange the plots, click Window, then Tile Special, then Large (or adjust
tiling strategy as desired).
10. Basic regions can now be created using icons from the toolbar or by clicking
Analysis, then Create Region and then choosing the desired region. You must
first click on the plot upon which you would like to create the region before you
may click on an icon from the toolbar.
11. To apply a region as a gate on a plot, right-click on the plot, then select Format
Plot. In the Data Source tab, select the appropriate region from the Gate drop-
down. If you wish to apply the region as a gate to multiple plots, the fastest way
is to check Apply gate to all plots. To remove the gate from plots that do not
require the gate, right-click, select Format Plot, and change the Gate drop-down
to Ungated.
12. To create an automatic stop when a certain number of cells are displayed on a
specific plot, right-click on the plot, then select Format Plot. In the Stop and
Save tab, check Use Stop Condition, then enter the desired number.
13. Additional or fewer statistics can be reported. Select Analysis, then Select
Results. Select the statistics that are desired.
14. Appropriate voltages and compensation should be determined for each parameter
using a sample of unstained (or otherwise non-fluorescent) particles, as well as
the required controls (see section on Running Samples, Single Tube mode).
Regions should also be adjusted. Save the protocol after any change is made!!!