LSR Fortessa X20 | Reservations

Optical configuration

  • The X-20 is equipped with 4 different low-power lasers that can be used simultaneously. A 50mW solid-state blue (488nm) laser, from which SSC and 2 colors can be detected; a solid-state 40mW violet (446nm) laser with 6 detectors, a solid-state 40mW red (446nm) laser, from which 3 colors can be detected, and a 40mW yellow-flowcytometry (561nm) laser where 5 colors can be detected. Instead of calling the detection channels generically FL1-to-FL16, each channel is named after it's respective PMT detector, to easily setup an experiment users should label which fluorochromes are being detected. Note that a band-pass filter designated as 530/30 indicates that it will detect light in the a 30nm range centered at 530nm, i.e., 515-545nm.

Operating Procedures

  • Before Acquisition

    1. If machine is turned OFF, follow the Startup LSR X20 procedure.
    2. Check if sheath tank is full. If necessary refill tank with PBS.
      Clean up all spills. PBS is corrosive!
    3. Start BD FACSDiva and log in as USER (if you require credentials ask the UIC staff).
    4. Wait for the cytometer to connect to the computer.
    5. Remove tube from Sample Injection Port (SIP) and PRIME 3 times (with support arm to the side)
      Check Bubble Filter and flow cell forbubbles. If necessary slightly tap the filter while opening the bleed valve, 1st the one connected to the orange quick-connect then the other.
    6. Run PBS for 1 min in HIGH to check if machine is dirty. If necessary follow the Cleaning Procedures.
      Though events may appear, it may not be due to poor cleaning and they may or not interfere with acquisition. Evaluate noise events based on the location and event rate during sample acquisition.
    7. Open/Create your Experiment and setup acquisition plots in FACSDiva.
    8. If CST Mismatch notification shows up select: Use CST Settings
    9. You are ready to start running your samples.
  • Startup Fortessa

  • After Acquisition

    1. Run Cleaning Procedures
    2. Export your acquisition files from FACSDiva to:
        D:\EXPORT – Folder routinely cleared and REMOVE all your data from the software and hard drive. (For more information see Saving and transferring Data section)

    3. When finished, leave the X20 on Standby with dH2O on the SIP and refill the sheath tank. Check if there is a user reserved after you in the next 3 hours.
      IF YES: leave the X20 on Standby. Close FACSDiva software and Log Off from Windows session.
      IF NO: Switch OFF X20. Close FACSDiva software and Log Off from Windows session.
    4. Export your acquisition files from Diva and REMOVE all your data from the software and hard drive.
      The computer is NOT for data storage.
      The hard-disk is subject to routine cleaning, and files may be deleted without warning.
      Suggestion: You may remove your files while running Cleaning Procedures (see instructions on how to transfer files to the xservers).
    5. Remove your gloves, tubes and other belongings and clean up any trash.
    6. If you have any doubts or need assistance call the facility's staff (ext. 251).
  • Cleaning Procedures Fortessa

  • Trasfering Data

    Acquired data is saved directly on the hard drive. Use the D:\EXPORT - Folder routinely cleared\ partition for this purpose.

    The way data is exported, depends on where the data is to be analyzed:

    • ![endif]>export as Experiment if you are going to analyze on an offline FACSDiva software & wish to keep plots & gates

    export as FCS files to analyze data in any flow cytometry software

    To avoid reaching the limit size of FACSDiva Database file, all Experiments must be exported after acquisition. Please confirm if files were successfully exported to your folder and DELETE them, leaving only an empty Experiment template within the software.

    Due to space constraints, all data is routinely cleaned. To avoid data loss, the users MUST transfer their data to another location. The best way to store your own data is to transfer it to a pen-drive, USB external hard disk, or to your directory in remote server. To transfer data to filespc or to another network location, follow these steps:

    1. Open Filezilla software.
    2. You'll be prompted for your username and password.
    3. Select the folder you want to access.
    4. Locate and drag your local files into the remote folder.
    5. To disconnect from the remote drive just close Filezilla.

  • Custom Changes

    The machine also includes a direct waste system eliminating the need for a waste tank. Therefore, users do not have to keep changing the waste tank when it's full. The sheath tank, however, must be ALWAYS refilled after usage, or if running low on PBS.