IGC

BD FACSAria IIu | Reservations

Optical configuration

  • FACSAria IIu is equipped with 3 different low-power lasers that can be used simultaneously. A 13mW solid-state blue (488nm) laser, from which SSC and another 5 colors can be detected (upgradable to another 2); an air-cooled 11mW HeNe red (633nm) laser, from which 2 colors can be detected (upgradable with an extra color); and a solid-state 10mW violet (407nm) laser, from which 2 colors can be detected (also upgradable with an extra color). Instead of calling the detection channels generically FL1-FL9, BD named each channel after the most typical fluorochrome it detects (see picture below). 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. LP stands for Long Pass filter, and indicates that it will detect light above the mentioned wavelength. A list of typical fluorescent labels detected in each channel is also displayed on the right.

Operating Procedures

  • Startup FACSAria IIu

    1. If you're going to sort, turn cooling bath ON. If not, leave it OFF.
    2. Lift flow cell access door.
      This avoids having the lasers burning possible dry PBS crystals in the flow cell, and damaging it.
    3. Turn ON the FACSAria IIu (flowcytometry button on the left panel).
      Make sure that at least the BLUE laser is ON before switching on the machine , otherwise the machine won't acquire (Bug reported by BD).
    4. Turn ON the remaining lasers that you'll need for acquisition.
    5. Launch FACSDiva software.
      Wait a couple of minutes while the system connects.
    6. Make sure electric plates are clean and dry, and adjust the angle.
      Plates should not be too close to each other to avoid sparking and damage to the electronics.
    7. Check fluidic levels in the Instrument window.
    8. Once the instrument is connected, from the Instrument menu run Fluidics Startup.
      Instrument replaces H2O, left during last shutdown procedure, with PBS.
    9. Start the stream.
      Make sure stream is falling in the center of the waste tube. If not, adjust accordingly.
    10. Close the lid to open the laser shutter.
    11. Open/Create an Experiment and setup acquisition plots.

  • Shutdown FACSAria IIu

    1. Unload sample tube, if one is loaded.
    2. Turn OFF red and violet lasers.
    3. Open the flow cell access door.
    4. Turn stream OFF.
    5. Choose Instrument->Fluidics Shutdown.
      Follow the indications provided by the software.
    6. At the end, you're prompted to "Install a tube with cleaning solution on the loading port, and click OK". Install a tube of dH2O and select OK.
    7. Switch OFF the instrument (flowcytometry button on the left panel).
    8. Close FACSDiva Software.
    9. Clean waste tank (see Cleaning Waste Tank).
    10. If you were sorting and using the temperature bath, switch it OFF.

  • Cleaning Waste Tank

    After switching off the instrument, wipe every surface that has been exposed to sheath fluid, to prevent salt build up.

    Cleaning the Waste Tank:

    1. There are two waste tanks: One with waste, and the other one empty (with a bit of bleach only). Unscrew waste container's sensor from the filled tank and the top of the empty one.
    2. Tip the full tank slightly, remove the sensor, and screw it back in the empty tank.
    3. Remove the large-sized red cap from the container, dispose of waste, and rinse twice with tap water.
    4. Add approximately 100 ml of bleach to the waste container (10 liter container).
    5. Close the waste container by screwing back the large-sized red cap, and the white small cap.
    6. Place the tank back in the fluidics cart, changing places with the empty tank.

  • Saving and transferring Data

    FACSAria is connected to a PC loaded with BD FACSDiva Software for machine interface and data acquisition. Whether you're sorting or acquiring, FACSDiva Experiments are saved within the actual software (in one single database file, containing all the other saved experiments). To avoid reaching the limit size of this database file ~16GB, all Diva Experiments should be exported, and after confirming there is no data loss, they should be deleted from within the software. The way the data is exported, depends on where the data is to be analyzed. If you wish to maintain the same plots and gates drawn in the FACSDiva software, you must export the actual Experiment and then you may import it back in any offline FACSDiva software. To analyze the data, for instance, with Flowjo, data must be exported as FCS files (the "Flow Cytometry Standard"). Depending on the software and its version, you may have to choose FCS 2.0 or FCS 3.0.

    Once data has been exported to the hard drive transfer it by saving it to a pen-drive or transferring it to your directory in xserve02. To transfer data to xserve02, follow these steps:

    1. Click on one of the xserve shortcuts on the Desktop.
    2. Enter username and password.
    3. Select the folder you want to access.
    4. Drag your local files into the remote folder.
    5. To disconnect from the remote drive you have to run one of the 'windows-logout' executable files located in the remote servers.

    IMPORTANT: After you have checked there is no data loss in your exported experiment, delete the Experiment from the FACSDiva Software. FACSDiva software is prone to crashes and "mysterious" data losses have been reported by the flow cytometry community. The first thing to do after any flow analysis or sorting, is to save your own data.


  • Custom Changes

    Though we don't have any real custom changes in the FACSAria, we do have some optional components:

    1. BD Temperature Control Option. This includes a recirculating water bath for temperature control of the collection tubes and/or ACDU stage, and specially designed collection tube holders for 15 ml two way sorting and 5 ml four-way sorting.
    2. BD Aerosol Management Option (AMO). This includes an attached vacuum source to rapidly evacuate aerosolized particles that go through an ultra-low penetrating air (ULPA) filter to trap these particles.
    3. External air supply to pressurize the instrument. This is connected to the Auxiliary Air Supply connector in the fluidics cart, giving a constant pressure of 85 PSI.

  • Sample Preparation

    1. Human samples must be serology tested (HIV and Hepatitis B negative) before brought to the Flow Sorting room.
    2. Samples must be single cell suspension (SCS) only (i.e. no clumps).
    3. To prevent clumping during the sort, filter the SCS through a ~30µm mesh
      • We suggest to use the 5 ml Polystyrene tube with Cell-Strainer Cap, Falcon #352235, or Pre-separation filters.
        Filters are also provided by the facility.
      • You can also add 0.5 mM EDTA to your buffer solution;
      • Use 1-5% serum for the buffer.
    4. All samples can be placed in the following types of tubes:
      • 1 ml microtubes, Bio-Rad Laboratories, #223-9391.
      • 5 ml polystyrene and polypropylene test tubes, 12 x 75 mm (BD FalconT):
        • Non-sterile, #352008;
        • with Cell-Strainer Cap, #352235;
        • Capped, #352005, #352054, or #352058;
        • Uncapped, #352052.
      • 15 ml conical polystyrene or polypropylene centrifuge tubes (BD FalconT)
    5. Please provide the controls - unstained and single color controls. Preferable concentration of the controls is around 1 x 10^6cells/ml, volume 0.5-1.0 ml.
    6. The sample concentration for the sort should typically be around 10-15 x 10^6cells/ml. If you have fewer than 5 x 10^6 cells put them into a maximum volume of 1 ml.
    7. Please provide extra 15 ml of the buffer you use for the cell samples.
    8. Please provide collection tubes for the purified (post-sort) cells.
      • For the post-sort tubes use eppendorf tubes, any type of 12 x 75 mm tubes (made of glass, polypropylene, polystyrene, etc.), or 15 ml Falcon centrifuge tubes;
    9. In order to prevent cells sticking to the sides of the tubes, pre-coat the tubes, filling them with the serum for 30 minutes before the sort;
    10. For sterile sort the post-sort tubes should also be sterile.