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Instrumentation

The Facility is equipped with a 2 BD FACSAria II SORP and 1 BD FACSCanto RUO Special Order. These instruments operate on a PC-based platform using FACSDiva software.

Analyzer

FACS Canto RUO Special Order

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The FACSCanto RUO Special Order is equipped with three lasers (405nm, 488nm, and 633 nm) that offer simultaneous 12-parameter detection and a FACSLoader. This state-of-the-art instrument offers fixed optical alignment as well as automated compensation and housekeeping routines making it highly desirable to operate.

    1. Titrate antibodies. Antibody titration is a quick experiment designed to find the optimal staining concentration for the antibody being used on the cells of interest. This ensures saturating your cells with the antibody, and not having excess that will lead to reduced resolution because of non-specific binding (NSB).
    2. Become a fan of the FMO controls. Setting proper gates is critical for good cytometry. One of the critical controls for doing this is the fluorescence minus one (or FMO) control. The FMO control allows for the visualization of the spread of the data due to the other fluorochromes in the panel. It is especially critical when you wish to make a sensitive measurement or for rare events.
    3. Optimize instrument voltages. If embarking on a long-term project using the same staining panel, it is worth the time to ensure that the instrument is giving you the best sensitivity for the cells. Use the BD Application Setting on DiVa for this purpose. (Ask CRI Flow staff for more details or help.)
    4. Value the viability dye. Dead cells will non-specifically take up all antibodies in the solution and can appear to be positive cells, thus confounding the results and counts. To combat this, add a viability dye. From cell impermeant dyes like PI, DAPI and 7AAD to the amine reactive dyes, the myriad choices will fit your assay conditions and your instrument configuration. Viability dyes can help combat non-specific binding.
    5. Adopt the median. There are three main ways to report the central tendency of a population. For fluorescent flow cytometry data, adopt the median. The median is the most robust measure of the central tendency on flow data for several reasons:
      • The median does not require all the values to be known, so if some are off scale, a median can still be calculated.
      • The median does not assume the data fits a specific model (i.e., the normal or Gaussian distribution).
      • The median is resistant to outliers.

Cells Sorter

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FACS Aria II SORP (4 lasers and 5 lasers)

The FACSAria II SORP offers state-of-the-art features in the most advanced cell sorter currently available on the market.

The FACSAria II SORP has air-cooled lasers and fixed-alignment cuvette flow cell, which allows achieving superior fluorescence sensitivity. BD FACSAria II delivers high-speed multicolor sorting with digital acquisition rates of up to 20,000 events/second. It is able to achieve sort into two- and four-way bulk sorting devices for a variety of tube sizes, or can sort using Automated Cell Deposition Unit (ACDU) to multi-well plates or microscope slides.

Two FACSAria II SORP are available in the Facility:

  • Four-laser system (405nm, 488nm, 561nm, 633nm), which simultaneously collects multiple emission wavelengths (up to 13 fluorescents detectors)
  • Five-laser system (305nm, 405nm, 488nm, 561nm, 633nm) that accommodate up to 17 parameters -15 colors and 2 scatters

The two FACSAria II SORP have been placed in a class II biosafety cabinet designed to provide personnel, product, and environmental protection from potentially hazardous, aerosolized particulates.

Your sort will go far more quickly and efficiently if you read these instructions beforehand, as the Flow Core facility strictly adheres to these guidelines:

  1. Viable cells should be re-suspended in a low protein buffer for sorting. High protein concentrations can disrupt sort stream formation. We typically use 2% FBS in Ca/Mg-free PBS with 0.5 mM EDTA, although any variation on this (BSA, RPMI, HBSS) will generally work. Protein additions and media should be sufficient to keep the cells alive for the duration of the sort. Sorting adherent cells adds a level of complexity to an experiment. The cells have to be disassociated to pass through the sorter, and this is often done with trypsin. The quickest and most common neutralization method is to add FBS to the cells.  Be careful of this; while it neutralizes the trypsin effectively, it also adds back all the components that cells need to re-adhere to each other. Try using soybean trypsin inhibitor instead. You might also want to include a viability dye in your staining panel. This will help eliminate dead cells. Using a viability dye is always a smart decision.
  2. Cells should be filtered through nylon mesh (70 microns maximum) immediately prior to sorting to prevent nozzle clogs. If your cells are particularly susceptible to clumping (as are many adherent cells), sorting the cells in Ca/Mg-free buffers, adding 0.5 mM EDTA and DNAse may reduce aggregate formation, which can clog the sort nozzle and impede sort performance.
  3. The FACSAria II SORP can accommodate 1 ml micro-tubes and 15 ml conical tubes in addition to the 12×75 mm Falcon (Becton-Dickinson) polystyrene tubes. Know the cell count at the time the cells are going onto the sorter – not from when you first began preparing them. Since an optimal sort speed is typically ¼ the droplet generation frequency, over-concentrating the cells will reduce purity at the back end. Bring some dilution buffer with you in case the cells are too concentrated. Cell concentration should be no more than 30×106 per ml for the FACSAria II SORP.
  4. Sorted cells are collected into tubes or plates. Collection tubes may be eppendorf tubes, ppn tubes, 12 x 75 mm round bottom or 15 ml conical tubes. Cells are going to be traveling in a buffered saline. This is not very conducive for keeping cells alive for long periods of time. The good news is that you can improve your recovering by ensuring that the catch buffer has some – but not too much – protein in it. Typically only 10-50% protein in the catch buffer is sufficient. To get better recovery, pre-coat (incubate your plastic tubes with a buffer solution containing protein) the tube with protein/buffer to neutralize the plastic charge. Even better, make sure that your tubes are polypropylene, as it is less charged than polystyrene, thus reducing “droplet spray” and tight adherence of the sorted cells to the walls of the tube. The FBS also reduces cell adherence to the walls of the tube and provides a “cushion” for the cells when they “land.” A sufficient number of these tubes should be prepared beforehand and delivered with the cells to be sorted. If you’re sorting into media, make sure the media is HEPES buffered.  Buffers like RPMI are formulated to buffer in a CO2 atmosphere (like the atmosphere found in your lab’s incubator) and, as such, don’t buffer well in our normal atmosphere. Plates can be between 6 to 384 wells plate.
  5. The FACSAria II SORP is equipped with a temperature-controlled sample chamber and collection tube holder to keep both pre- and post-sorted cells at 4°C or 37°C. Tell us in what temperature you need your cells to be sorted.