Cell Sorting Core Facility

Services

Routine Services

The three flow cytometer/cell sorters in the core facility are equipped with dual lasers, forward angle light scattering detectors, 90Ą light scattering detector and four PMT tubes for multi-parameter analysis and cell sorting. The routine services include:

Immunofluorescent measurements (surface and intracellular markers, single and multi-parameters) for antigen and antibody distributions.
DNA, RNA, and nuclear antigens for cell cycle, apoptosis, and cell proliferation (BrdU) analysis.
Sterile cell sorting and auto-cloning based on light scatter and fluorescent intensities (antibodies, DNA, RNA, GSH, GFP, etc.)
Measurement of green fluorescence proteins (GFPs) for analysis and cell sorting.
Bulk cell separation by centrifugal elutriation for large quantities of host and malignant cells, and cells at different phases of the cell cycle.

Developmental Research

The core assists users to adapt and modify existing flow cytometric methods or to develop new procedures for their specific requirements.

Training and Education

The core offers both formal and informal training to users who are interested in the theoretical and practical aspects of flow cytometry and cell separation technology.

University courses
Instrument operation
Analysis software

Protocols for Sample Preparation

As a general requirement, all samples should be prepared in a single-cell suspension form at a concentration of 1-2 x 106/ml. Samples should be filtered through a 35-40 m filter to remove any clumps or aggregates prior to analysis.

Fee Schedules

A fee structure is employed to determine the charges for the usage of the Cell Sorting Core. The structure is based on the average charges posted by other institutions in our region, and on the capability of individual instruments in the Core.

Analysis by users:
FACS Calibur	$35.00/hr (to the nearest 1/2 hr)
Elite ESP	$35.00/hr (to the nearest 1/2 hr)
Analysis by Core staff:
FACS Calibur	$45.00/hr (to the nearest 1/2 hr)
Elite ESP	$45.00/hr (to the nearest 1/2 hr)
A charge of 1/2 sign-up time will be billed for late cancellation (<24 hr) or if less than 1/2 sign-up time is used.
Cell sorting:
FACS Vantage	$45.00/hr plus 1hr for non-sterile set up or 2 hr for sterile set up
Elite ESP	$40.00 /hr plus 1 hr for non-sterile set up or 2 hr for sterile set up
A charge of 4 hr sorting time will be billed for late cancellation (< 2 days) if the time cannot be reassigned.

Sign-up Procedures and Turn Around Time

The basic sign-up procedures and the first come-first serve policy for any of the analytical instruments will remain the same as before. Users will be able to sign-up for any analytical instrument up to 2 weeks in advance and for no more than a 3 hour block. Advanced sign-up longer than 2 weeks or a block of time longer than 3 hours requires prior approval from the Core Director. Any cancellations must be made at least 24 hours prior to the sign-up time or you will be billed for the time that is reserved for you.

Sign-up and scheduling for sorting experiments should be discussed with the Core staff first because often time the experimental details need to be sorted out before an exact amount of time can be assigned. Currently, advanced sign up time for sorting is 4 weeks due to heavy usage of the FACSVantage sorter. Please note, cancellation by e-mail for cell sorting, should be made at least 2 days prior to the scheduled time in order to reassign the time slot to other users.

Training Courses

A year round training schedule is established by the Cell Sorting Core to provide continuous training on the basic and advanced flow cytometry techniques for both existing and new users.

The training classes will be given on the first Monday afternoon of each month with basic training offered even-numbered months and advanced classes during odd-numbered months. The basic training course will include a one-hour lecture on the principle of flow cytometry and in addition a two-hour hands-on section on software and operation. The advanced courses will include principles and hand-on experiments for cell cycle analysis, digital software, and cell sorting. ALL new users including technicians, graduate students, and postdocs are required to take the basic training course before they are certified as qualified users. Each individual will be charged $105.00 per session and this amount will be applied to the first three hours of instrument usage. The training course will start in February 2005. If you are interested in attending the training courses, please send your request by e-mail to peter_keng@urmc.rochester.edu

When: Monday afternoon 2:30 - 3:30 PM (lecture), 3:30 - 5:30 PM (lab)
Where: Cancer Center conference room G-3111 (lecture), 3-4148 (lab)

February 7th	basic training
March 7th	cell cycle analysis
April 4th	basic training
May 2nd	DiVa digital software
June 6th	basic training
July 11th	cell sorting
August 1st	basic training
September 5th	cell cycle analysis
October 3rd	basic training
November 7th	DiVa digital software
December 5th	basic training

Literature Citations Related to Core Methods

Robinson J.P. Handbook of Flow Cytometry Methods, Wiley-Liss, New York, 1993.
Shapiro H. Practical Flow Cytometry, Third Edition, Alan R. Liss, New York, NY, 1994.

In addition, we have prepared a handbook that covers most of the commonly used staining and analysis procedures that are currently employed in the core facility. Advice on necessary controls, trouble shooting, and common staining procedures are available. Please contact the core staff for details.

Literature and Procedures

Basic Training

Contact Information

Peter_keng@urmc.rochester.edu

Other individuals to contact

Tara Calcagni, 275-6330
Michael Strong, 275-2763