University of Pennsylvania

School of Dental Medicine

Flow Cytometry Facility

Introduction

FACS User Committee

• Dr. Bruce J. Shenker
• Dr. Gary Cohen
• Dr. Edward Lally
• Dr. Ellis Golub
• Dr. Joel Rosenbloom

Guidelines for using the FACS

• FACS use must be approved by the Users Committee when:
• Any project is being initially considered for the cell sorter.
• Demand for the cell sorter exceeds available time.
• Criteria for determining FACS use priorities:
• Is the project within the capabilities of the FACS as configured?
• Does the project require methodology unique to the FACS or can the project be completed using other available techniques?
• Will the project clarify the effectiveness of other separation methods which are able to process larger numbers of cells than the FACS?
• Is the investigator a member of the SDM?
• Biological hazards and non-physiologic media:
• Cells which are radioactive or are shedding virus will not be accepted without specific approval of the FACS coordinators.
• If an investigator suspects the presence of any pathogen or carcinogen in a cell preparation use of the FACS must be discussed with the FACS coordinators.
• Since non-physiologic media may damage the FACS or cells subsequently passed though it, such media will not be accepted without approval of the FACS coordinators.
• Investigator's Responsibilities:
• For each/new project submit a brief statement describing its objectives and the need for the FACS to reach them.
• Specific experimental objectives and protocols should be discussed with the FACS coordinator Dr. Bruce Shenker.
• Arrange for FACS time with the operators Mark Miller or Sugandha Datar. Since FACS time is assigned primarily on a "first-come first-choice" basis it is to the investigator's advantage to sign up at least one week in advance. Advance notice is especially important for sterile sorting and/or reconfiguration of the lasers.
• Cells should be provided in 12x75 mm plastic test tubes (Falcon #2054) in appropriate media and concentration. To some extent cell concentration will be dependent on type and tendency to aggregate. In general, cell concentrations should be at least 10⁷ cells/ml for sorting and 10⁶ cell/ml for analyses in minimum of 0.3 ml.
• Provide a data sheet for each experimental run containing the following information for each tube: cell type, probes used, and any prior manipulations (e.g. panning, rosetting, etc.)
• If cells are sorted, submit a summary of consequent use and results to the FACS coordinators, especially following a "sterile" sort.
• Submit bibliographic information on any publications containing data obtained on the FACS. If reprints are available, please include one. This information will be beneficial to the continued financial support of the FACS facility.
• Hard-Copy Data: Computer printouts of FACS data will be available to the investigator as soon as possible, usually no later than the day after data acquisition. Several programs are currently available for data reduction and the library will be expanded as needed. Permanent records are kept of all FACS data and on request can be used to generate customized hard copy suitable for reproduction as slides and/or manuscripts.

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Request for FACStar Plus Access

1. Cell source and type (e.g. mouse spleen, rat T cell line, etc.)
2. Initial cell purification procedures (e.g. erythrocyte lysis, trypsinization, collagenase treatment of solid tissue, etc.)
3. Type, purity and previous assay method of fluorescent reagents.
4. Expected frequency of FACS access.
5. If cell sorting (as opposed to analysis alone) will be performed, will sterility be required?
6. Sorting criteria (i.e. purity, number and type of cells required after separation.)