Sample Preparation for Sorting
Sample Preparation for Sorting
Your sort will go much more quickly and efficiently if you read the following instructions beforehand, as the Flow Cytometry Facility strictly adheres to these guidelines.
- Viable cells should be resuspended 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 0.5mM 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 to use 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.
- 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 0.5mM EDTA and DNAse (25-50µg/ml) may reduce aggregate formation which can clog the sort nozzle and impede sort performance.
- The FACSAria II SORP/FACSAria Fusion 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 just in case the cells are too concentrated. Cell concentration should be no more than 30×106 per ml for the FACSAria II SORP/FACSAria Fusion SORP.
- 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, 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.
- Temperature control.The FACSAria II SORP and FACSAria Fusion 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 42°C. Tell us at what temperature you need your cells to be sorted.