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FACS Core Facility  / Technical Considerations

Technical Considerations

Core employees are available for advice on sample preparation and some reagents are available for testing. In particular, for indirect staining we have several cell surface molecule-reactive monoclonal antibodies available in the Medical Research Center (Moab_data-sheets_Bühring.zip). Many of them are clustered (CD asssignments; CD1-350) www.hcdm.org

Data analysis is available on work stations located at the Core Facility. However, it is recommended that users organize their own data management. There is current discussion whether to use Flowjo software from TreeStar Corp. for analysis, FCS Express from De Novo Software, or free analysis software programs available that can be installed on any computer (i.e. WinMDI, Joe Trotter). A tutorial for WinMDI is available from the Purdue university.

All samples must be in 12 X 75mm polystyrene tubes only from Falcon. In general, cell densities should be 1x106 to 1x107/ml, and each tube should have a volume of 300 μl to 3 ml.

ALWAYS bring NEGATIVE AND POSITIVE CONTROLS. To make appropriate conclusions about your samples the proper controls are necessary. Negative controls: Cells only (No stain). Specificity controls: If an indirect antibody staining method is employed, it is important to include a control in which cells have been stained with only the secondary antibody. This will indicate if there is "non-specific staining" or Fc region binding from the secondary antibody. Compensation controls: In experiments which require simultaneous staining using two or more fluorochromes, it is necessary to prepare controls that have been stained with each dye separately. These single color controls are required for adjusting cross-over signals between dyes or detectors and are crucial for multicolor immunophenotyping.

Samples which are suspected of having aggregates (cell clumps) must be filtered (we recommend filtering of each sample). 40 micron nylon mesh is available either at the Core Facility or can be purchased by Essrich & Weise. Falcon brand cell strainers for 50ml conical tubes (Falcon #35 2340 - 40um and Falcon#35 2350 - 70um) and filter top caps found on Falcon brand polystyrene 12x75 mm tubes (Falcon #35 2235) can also be used. We also recommend samples should be kept on ice or chilled until ready to run on the machine.

PLEASE SHOW UP ON TIME. You should give at least 24 hours notice if you need to cancel an appointment. If you are late for an appointment and run into the next users appointment you may either be excluded from sorting or have the samples sorted at a later time. In particular, if you are going to be late for a sorting appointment more than one hour then it is your obligation to check to see if your sort can still be done because other sorts may be scheduled on the same day. If there are other sorts scheduled then they will have priority.

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