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Beckman Coulter Cytomics FC 500 User Manual
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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!!!

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Summary of Contents for Beckman Coulter Cytomics FC 500

  • Page 1 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.
  • Page 2 15. Select the appropriate Flow Rate from the drop-down menu (recommended: Low or Medium). Changing Axis Name Purpose: It may be desirable to change the word(s) on an axis. The steps below describe how to input the axis name so that it shows on the graphs and gets stored in the LMD file.
  • Page 3 b. Click on the plot and hover the mouse over the axis name until “>>>” is displayed. Click. *In the window that opens, click on the Labeling tab. *Find the appropriate axis (X-axis or Y-axis) and select $Stain from the dropdown list. *Click OK.
  • Page 4 Naming LMD Files Purpose: At each use of the software, adjustment of the User’s settings controls how LMD files will be named. This procedure describes multiple ways to set up the Workspace Preferences to achieve the desired naming strategy. A. General Guidelines 1.
  • Page 5 2. If running in Single Tube mode, set Workspace Preferences (see A1 above) as follows i. In LMD File Name tab, uncheck all boxes except for Sample ID1 1. If a different component that is not manually entered for each sample (i.e. Run #) is desired, check that desired component instead of Sample ID1 ii.
  • Page 6 Running Tubes A. Single Tube Mode allows the user to place one tube at a time onto the carousel. The sample is run, then the instrument stops & returns the tube. The first tube must be removed and then another tube can be loaded in the same position. All tubes are placed in position 10 of the carousel, which can be accessed by the small Tube Access door on the front of the carousel lid when the instrument is in Single Tube mode.
  • Page 7 B. Automatic Mode allows the user to load up to 32 sample tubes into the carousel and program the instrument to automatically run each tube in sequence. Automatic Mode is the default setting for the CXP Software; no special button must be pressed to enable Automatic Mode.
  • Page 8 Quality Control check on the FC500 Every morning (Monday - Friday), Facility staff run 2 QC bead sets to ensure that the lasers & PMTs are functioning normally. The QC beads also check to make sure the lasers have "warmed up" enough and are functioning at the appropriate power level. Very rarely do we see results that are abnormal - and usually when we do the problem is either a) a bubble in the fluidics (solved by priming several times)
  • Page 9 8. After running the FlowCheck 9434031 beads, find another tube in the refrigerator labeled "675 beads 763253." Open the "1A_675 Beads 763253" Acquisition Protocol under FLOW. Be sure that the numbers on the tube match up with the numbers in the Acquisition Protocol.
  • Page 10: Setting Printing Options

    Setting Printing Options Purpose: Turn on/off printing of FlowPages. Some users wish to have these printouts; others do not. 1. Printing preferences are controlled in the Workspace Preferences. To get to this, click File, then Workspace Preferences, or press CTRL+W. 2.
  • Page 11 Start-up Procedure: 1. Verify that the Sheath Fluid container is full. Fill with Sheath Fluid to the blue line. 2. Verify that the waste container is empty. If it contains waste, carefully dump into sink and add bleach to the first line (bleach is under sink) 3.
  • Page 12 Routine Cleaning for All users after each use: (please note: feel free to log out of RIMS before washing and/or shutdown – you should only be charged for time you spend with your research, not cleaning!) 1. After finishing your data acquisition, clear the worklist by clicking should see the Acquisition Manager clear.
  • Page 13 9. When the cleaning panel is finished, the messages at the bottom of the software window will read “Awaiting Sample” and “Set Automatic Mode” 10. Remove the bleach and water tubes from the carousel 11. If shutting down instrument, proceed to Shutdown Procedure instructions. Shutdown Procedure: (all after-hours users should shut down the instrument after cleaning unless the next user is in the room)
  • Page 14 by finding your folder and data in the “Users (FC500)” folder (Should be under DesktopFC500 UsersPI’s FolderFCS filesyour data folder) 6. You have now transferred your data, you can log out and access it from your own computer by going back to webfiles.psu.edu (or your storage website)