University of Pittsburgh Cancer Institute (UPCI)

CCSG Acknowledgement

Required CCSG Acknowledgement

The NCI requires that publications acknowledge the UPCI CCSG support, and they are tracking compliance. If a UPCI CCSG-supported Shared Resource provided data used in your publication, please include the following statement in the acknowledgment section of your publication(s):

"This project used the UPCI [insert name(s) of shared resource(s)] that [is/are] supported in part by award P30CA047904."

Shared Resource Directors: Please make sure to include this statement on all of your order forms, contracts, etc. as a reminder to your users to acknowledge the UPCI CCSG support.


Sorting

Cell Sorting or FACS (Fluorescence Activated Cell Sorting) can be used to isolate high purity fractions of cells in suspension. The cell sorter combines multi-parameter flow cytometry with the ability to sort individual cells into several distinct populations. Sterile, functional cells are collected and can be returned to culture, utilized in functional assays or further characterized through genomic, proteomic or similar analysis. Up to 4 populations, defined by Boolean gates, can be sorted simultaneously. Alternatively, a single population can be deposited directly into a variety of cell culture plate formats (e.g., 96-well plates). The number of cells/well can be specified for each well, facilitating cloning, single cell experiments or limiting dilution experiments, where graded numbers of cells are plated.

High speed sorting allows for the processing of large samples. With throughput rates up to 30,000 cells per second, millions of cells can be sorted in a very short amount of time. Cells can be sorted to optimize purity or recovery. The UPCI MoFlo high speed sorter is equipped with three lasers (UV, 488 and 633nm) and can utilize more than 10 parameters (8 colors) to identify specific functional or phenotypic characteristics and sort up to four separate populations simultaneously.

The fluidics components of our MoFlo are enclosed within a Class I Biosafety Cabinet and the laboratory is operated at a BSL 2+ level, allowing us to sort viable human and non-human primate cells safely. For information concerning sorting of potentially biohazardous samples, please see the BSL2 Safety Manual.

Instrumentation

Beckman Coulter (Cytomation) MoFlo High Speed SorterBeckman Coulter (Cytomation) MoFlo High Speed Sorter

The MoFlo high speed cell sorter is designed for high speed analyzing and sorting with capabilities to sort at up to 50,000 cells/second. It is equipped with two lasers (nine lines) and ten PMTs. It can be used for up to eight-color cytometry, simultaneously using as many as three laser lines with accessories of SortMaster Droplet Control, CyCLONE Automated Cloner and 4Way Sort System.

Helping to automate the sorting process is the SortMaster monitoring system. SortMaster automatically adjusts drop delay as it detects shifts in the sorting profile. These quick adjustments further enhance the probability of a successful sort. Four-way sorting increases flexibility and maximizes use of samples submitted for sorting. The CyCLONE Automated Cloner enables precision droplet deposition into a variety of microwell plate formats, slides or custom trays.

View fluorochromes for the MoFlo (requires Adobe Acrobat)

Special User Guidelines for the MoFlo High Speed Sorter

First time users or experienced users with a new project should contact Michael Meyer before setting up an appointment.

Sample Requirements

  • All samples must be in single cell suspension. Cells must be filtered through a <70 μm nylon mesh filter to prevent clogging the nozzle if the cell size is less than 70 μm.
  • Sample may contain 1-3% FCS, or <=0.2% BSA.
  • Cell concentration ideally should be at least 1x107 cells/mL and can be up to 5x107 cells/mL for non-adherent cells (low cell concentrations slow down sorting).
  • Sample must be in a 12 x 75 mm tube.
  • For the purpose of compensation for multicolor analysis or sorting, you must bring an unstained control tube plus single color stained tubes for each fluorochrome (>5% positive). Ask us about using IgG capture beads (BD CompBeads, Cat # 552843) instead of cells for single stained controls.
  • No radioactive material. The facility does accept unfixed human or non-human primate lentivirus-transfected cells, but investigators must meet with and discuss these experiments with facility staff. Please make an appointment if you need to perform a biohazardous sort.
  • Collection container (user supplies one of following options):
    • Tubes: 12 x 75mm, 1.5ml, 15ml or 50ml.
    • Plates:n24, 96 (include PCR plate), 384 well or custom size.
    • Trays/dishes or slides.
  • Fill collection container (~10% of volume) with desired collection medium.