IGC

BC MoFlo | Reservations

  • MoFlo is a High-Speed Cell Sorter produced by Dako Cytomation (now owned by Beckman Coulter). This particular model, manufactured in 1998, has analogue amplifiers and electronics (big electronic rack in the picture), and with the help of ADC's (Analogue to Digital Converters) is able to transmit data to a computer and where it is processed digitally. MoFlo, which stands for Modular Flow Cytometer, is highly configurable and adaptable to the researcher's needs. Our particular model is equipped with 4 lasers (>7 laser lines) and 9 photo detectors, enabling it to detect up to 9 colors simultaneously. MoFlo is serviced by our own facility, and we have made several modifications described below. Typical fluorescent labels detected are Hoechst, DAPI, CFP, FITC, Alexa488, GFP, CFSE, YFP, PE, PE-TexasRed, PE-Cy5, PI, 7-AAD, PE-Cy7, RFP, mCherry, APC, Cy5, APC-Cy7, etc). Only dedicated staff are allowed to operate the machine, and reservations both for analysis and sorting can be done by sending an email to the facility's staff. For more information, check the following topics:

  • MoFlo

Optical configuration

  • MoFlo is a jet-in-air system, and consequently sensitivity or excitation efficiency is lower than with cuvette-based flow cells (such as with FACSAria). This negative feature is essentially overcome by using higher power lasers, though some fluorochromes, such as PerCP and its conjugates, are not detected efficiently in this instrument. MoFlo is equipped with 4 different lasers, though only 3 can be used simultaneously. A 200mW Sapphire blue (488nm) laser, a Coherent I 90-4C 4Watt Ar-Ion laser (MLUV and several visible lines), a Spectraphysics 35 mW HeNe red (635nm) laser, and a custom mounted 50mW DPSS yellow (561nm) laser coupled to optics. The number of colors that can be detected by each laser depends on how the instrument is configured. It's therefore difficult to talk about a standard configuration, but we can display a typical configuration for two cases, e.g., when using the UV laser or the yellow laser, together with the other two (see pictures below). The several fluorescent channels are also termed FL1, FL2, etc... although their meaning is quite generic since they depend on how the machine is configured. Note that a band-pass filter designated as 530/40 indicates that it will detect light in the a 40nm range centered at 530nm, i.e., 510-550nm. DLP stands for Dichroic Long Pass filter, and indicates that it will transmit light above the mentioned wavelength and reflect the remaining light below that same wavelength in a given angle (MoFlos dichroics reflect in a 45º angle).

Operating Procedures

Learning to operate a MoFlo High-Speed Cell Sorter follows a steep curve, requiring a few weeks of dedicated training. For more information and the complete operating procedures for the MoFlo please check "For Flow Operators - Moflo" link.

  • Saving and transferring Data

    MoFlo is connected to a PC loaded with Summit Software for machine interface and data acquisition. Data files are saved as FCS files in the computer's hard disk. Files can be transferred to a pen-drive or to one of the xservers following 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: Remember to check if there is no data loss in your FCS files immediately after transferring the data. We are not responsible for any data loss occuring in MoFlo's workstation.


  • Custom Changes

    One of the main advantages the MoFlo is its open architecture allowing the core facility to adapt the instrument to the researcher's needs. It also allows us to implement technical modifications to improve the machine's performance:

    1. Fast Drop Delay Determination (FD3) System.
    2. Yellow (561nm) Laser coupled to Fiber Optics.
    3. Alternative and Interchangeable fluidic assemblies to sort either microorganisms or higher eukaryotic cells, avoiding cross contamination.
    4. Refrigirated 4-Way Collection Tube Holder.
    5. Custom Dichroic Holders.
    6. Separate PBS and dH2O fluidic systems for machine cleaning.

  • Sample Preparation Guidelines for Sorting/Acquisition

    1. Human samples must be serology tested (HIV and Hepatitis B negative) before brought to the Cell 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 MUST be placed in 5 ml polypropylene tubes, 12 x 75 mm (BD Falcon™), Capped (sterile) #352005, or uncapped (non-sterile) #352053.
    5. Please provide the controls - unstained and single color controls. Preferable concentration of the controls is around 1 x 106cells/ml, volume 0.5-1.0 ml.
    6. The sample concentration for sort should typically be around 10-15 x 106cells/ml. If you have fewer than 5 x 106 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.);
    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.