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Laboratory Protocols

Sample Presentation

  • For Flow Cytometry: Minimum volume is 0.25 ml, at a cell concentration of 0.5 - 2.0 x 10e6/ml (most commonly 0.5 - 1.0 ml at 1.0 x 10e6/ml). Cells may be resuspended in almost any medium, although PBS is preferred. Use 12x75 plastic tubes, preferably Falcon 2063 polypropylene tubes (Falcon 2063 tubes required when using the autoloading carousel). Note that tube dimensions vary slightly between manufacturers, so not all brands of tubes will work. Also, while polystyrene (clear plastic) tubes may work, the sometimes have barely visible cracks which can prevent the tube from being properly pressurized to maintain flow.
  • For Cell Sorting: Cells should be at a concentration of 5 - 20 x 10e6/ml in PBS or other suitable medium. Serum may be added up to 5%. For potentially sticky cells, supplement the PBS with EDTA to prevent clumping. Use only Falcon brand Falcon 352063, 352053, or 352002 tubes (12 x 75 polypropylene) as sample tubes. The collection tubes/plate will vary with the type of sort:  12 x 75 polypropylene tube, 1.5 ml microfuge, 15 ml or 50 ml tubes; or multi-well plates. A small amount of the appropriate culture medium should be added to each collection tube (e.g. 0.5 - 2 ml in 12 x 75 tube) or each well to help maintain cell viability upon sorting. Be sure to leave enough volume to receive the sorted cells, which will take up ~3 ml per million sorted cells using a 100 um nozzle. A sample preparation guide is available here.
  • Sample Filtration: Before cell sorting, all samples must be filtered through nylon mesh to remove large cell clumps and debris in order to prevent nozzle clogs. This is especially critical to avoid problems with cell sorting, since nozzle clogs can cause a significant delay as well as reduce the purity of the sorted sample. Use nylon mesh with openings ≤40 micron (e.g. Sefar Nitex, available from Small Parts, Inc. through Amazon). Falcon brand Cell Strainers (#352340) also work (available from Fisher Scientific).

General Protocols

FC-500 Daily Cleaning Procedure
The FC-500 flow cytometer must be cleaned after every use, without exception. Please be conscientious about cleaning; it will greatly reduce problems for all users. Click here for procedure: Cleaning the FC-500