Flow Cytometry Core Facility

INTRODUCTION

Flow Cytometry Facility
The Flow Cytometry Facility is a shared resource, which is available to all Cornell University researchers, and others. We are currently operational with two Beckman-Coulter XL flow cytometers, an Altra cell sorter, and a CompuCyte Laser Scanning Cytometer (LSC). The XL is a streamlined, user-friendly instrument and can be learned quite easily in a two-hour training course which is required for new users who wish to operate the instrument. Christopher Colon, the facility's Director, will be training and assisting all users. The Director is available to assist researchers in the development of flow cytometric assays and protocols for the preparation of samples, although it is required that all sample preparation be performed at the researcher's own facility unless otherwise arranged.

The facility is located at Weill Medical College at 1300 York Avenue, Room A-1027, (212) 746-6782.

Independent use

Users who have operated the XL analyzer instruments will be allowed unassisted use upon evidence of proficiency. Training in the use of the instruments is available on either an individual or group basis for a fee (see fee schedule) after which a user may be able to operate the instrument independently. The Altra cell sorter will be operated by core facility personnel only, with the requirement that the investigator be present for Altra sorting.

Assisted/ supervised use
The facility's staff or the Director will assist investigators in using the instrument based on availability, and will provide information for the preparation of samples for cytometric analysis. Samples must be prepared at the investigator's lab and must be in good condition prior to loading onto the instrument. Guidelines for sample preparation will be discussed prior to preparation, and well before presentation of the samples to the cytometers. If it becomes necessary to fully assist the user until s/he is able to be completely independent, then the charges that will apply are those listed for "Assisted Use" in the fee schedule below. The Facility is minimally staffed and does not accept samples for processing as a reference lab except by prior arrangement with the Director (LSC sample analysis may be an example of such an arrangement if the sample is in optimal condition; prepayment is required).

Assay/ protocol development
Flow cytometric analysis may not be appropriate for an existing preparation, in which case it will be necessary to develop a novel method of preparation or to research the literature for such a method. In these instances, the Director will be available for collaboration with the investigator. It is helpful for any investigator who wishes this type of collaboration to familiarize him or herself as much as possible with flow cytometric analysis and techniques prior to meeting with the Director. The publication, "Current Protocols in Cytometry" will be available on CD in the facility as a reference for assay development in flow cytometry. There are references for LSC sample preparation and analysis as well, however, this form of cytometric analysis being relatively new, may require that the investigator develop a unique sample preparation protocol.

Facility Description
The Flow Cytometry and Cell Sorting Facility is located at Weill Medical College, Room A-1027. The facility also has a Citrix server available for Cornell scientists who generate high volume data for analysis. Software is available for DNA cell cycle analysis, listmode analysis, and graphic display of data generated by most commerical flow cytometers (FCS format).

INSTRUMENTATION

Two Coulter XL analytical flow cytometers.
The XL is a Beckman-Coulter productwhich is designed for analytical flow cytometry and is easily operated with some basic instruction. The system features the capacity to analyze simultaneously up to four fluorescence wavelengths from fluorochromes which are excited by the instrument's air cooled argon laser (at 488 nm).

Laser Scanning Cytometer (LSC).
The Laser Scanning Cytometer (LSC) from CompuCyte Corporation is the latest instrument in the flow cytometry core facility. This instrument is used to measure fluorescence characteristics of cells on regular glass microscope slides, or their equivalent (75mm x 25mm x 1mm; read below for more information). Cells scanned with lasers are also available for visual inspection, unlike a flow cytometer. Less sampling is required than for flow cytometry and stabilized slides may be read repeatedly. The instrument has two air cooled lasers, an Argon and a HeNe. A violet laser will be added by the end of 2001.

Beckman-Coulter Altra cell sorter.

The Altra is marketed for the researcher who has the need to sort cell sub-populations. It is equipped with various lasers for the excitation of a wide variety of fluorochromes such as FITC, PE, PI, Texas Red, APC. The Altra is capable of sorting 10,000 events per second. This flow cytometer is a flexible and powerful tool intended for researchers who need the power and versatility not available with the analyzer model
above.

ORGANIZATION & SERVICES PROVIDED

Assay Development/Protocol Design
The investigator may ask for the Director's assistance in the development of novel assays/ protocols for a specific application, in which case a fee will be charged.

Flow Cytometric Analysis
Investigators are expected to have met with the Director of the facility prior to bringing any samples so that there will be as little sample loss as possible. There is no provision for the analysis of radiolabeled cells, or cells which have been physically treated with radioisotopes and which pose a potential radiation safety hazard. Also, preparations of potentially infectious materials must be fixed prior to analysis to reduce the risk of contamination. Cells and particles within the instrument specifications may be analyzed in concentrations of no less than 500,000 cells or particles per milliliter of suspension media. Typical analytes are mammalian cells at 1x10EE6 cells per ml. Other particles such as algae, microbeads, and aggregates or colloidal suspensions may be analyzed as long as they satisfy size limits (2-150 microns depending on other characteristics such as fluorescence intensity).

Laser Scanning Cytometry
The Laser Scanning Cytometer (LSC) from CompuCyte Corporation is the latest instrument in the flow cytometry core facility. This instrument is used to measure fluorescence characteristics of cells on regular microscope slides, or their equivalent (75mm x 25mm x 1 mm). The sample requirements are as follows (please call the director for more information prior to any sample submissions):

Cells must be free of one another and must be adhered to the surface of a regular, plain, glass microscope slide (or other optically clear, perfectly flat, non fluorescent, similarly sized material). Your cells cannot be moving!
Cells should be in a concentration which would allow for a mono-layer to be produced which will provide non-overlapping cells very close to one another, but not touching one another. There should be a stable fluorescent dye or fluorochrome present since this is a fluorescence-based technology (excitation wavelengths are from two lasers: Argon, HeNe).

Currently, the LSC instrument requires single-cell type preparations, not tissue sections (Compucyte has not fully developed its plan for this type of sample preparation).

This is an exciting and relatively new microscope slide-based technology which may require you to develop a new way of sample preparation for your own particular specimens. However, it is also one which would potentially grant you greater flexibility in terms of sample analysis than other methods currently available.

Because the cells are adhered to the slide and cannot move around, the LSC is able to record the position on the slide for each cell analyzed for possible later re-staining and re-analysis. The LSC provides data similar to flow cytometry data and which may be similarly manipulated. The advantage over flow cytometry is that one does not need as many cells, and that a sample may be analyzed more than once (data files may be combined for discrete runs of the same slide).

The following are examples of inappropriate samples:

Slides which are either smaller or larger than standard microscope slides (standard slides are 75mm x 25mm x 1mm).
Cells which are already fixed cannot be used (fixed cells do not stick or adhere to slides so that if you are going to fix your cells for staining it is best to do your staining on live cells, then allow cells to adhere to the slide for further treatment such as fixation and additional staining).
Cells which are overlapping and are crowded on the slide (cells in very low concentrations take longer to read and are difficult to set up. If you have a shortage of cells, you should attempt to concentrate them onto a smaller area on the slide using a cytospin prep if necessary, but remembering not to crowd them either!)

Training
Training in the use of the instruments is available to Cornell University users and for those who wish to familiarize themselves on the use and application of flow cytometry. This is a basic course that is intended for investigators who wish to operate unassisted. Also, basic instruction on software is available, although it is expected that each user will familiarize themselves with the software and will be analyzing data independently.

HOW DO I GET STARTED?

Contact the Director of the Flow Core Facility
Christopher Colon, 212-746-6782

Schedule a session
A calendar is available online for convenient scheduling upon which you must place your name, the desired instrument, your phone/extension, and time requirement, at least 24 hours in advance. Please contact the facility to arrange for your first appointment and to obtain the URL for the online calendar, as well as a username and password for future appointments.

Cancellation policy

XL and XLMCL analyzers:
You must cancel your reservation at least 24 hours prior to your reserved time by placing "CANCELLED" in front of your original reservation on the calendar. If you cancel more than 24 hours in advance you may delete your reservation completely from the calendar. Your account will be billed for the reservation unless you cancel at least 24 hours in advance in order to be fair to everyone, and to allow everyone to have equal access to the instruments (there are no exceptions; however, please feel free to come to the director if you feel you should be exempt from this policy). If the instrument is not functioning, you will not be charged for unused time.

Sorter and LSC:
You must cancel your reservation for the sorter or the LSC at least 72 hours in advance to avoid a charge, and to allow others to have ample opportunity to utilize the available time. The charge for cancellation will be a maximum of $125.00 regardless of the cause (avoidable, or unavoidable). As always, you will not be charged if another user makes use of your cancelled reservation.

Please note: If you keep staff over closing time during your session you will be charged at the assisted rate for the entire session. Please be on time and prepare to stop your analysis at least 10-15 minutes prior to your end time so that you have enough time to remove your data and can complete the instrument cleaning protocol.

Billing is as stated in the fee schedule.
ALL users must provide a valid account number prior to utilization of the facility.

Instrument/Service	Cornell Rate		Outside Rate
	Assisted	Unassisted	Assisted	Unassisted
Altra	75	N/A	150	N/A
XL	75	35	150	N/A
Data Archival Disks	20		20
Photo Quality Prints	10		20
Sorting	75		150
Laser Scanning Cytometer	125	90	250	N/A
Applications support/Assay development	125		250
Flow Cytometry Training Session	275		275

Rates/charges are per hour or any part of an hour. The facility reserves the right to change rates at any time without notice.

Notes:

1. Set-up time for sorter is charged at the hourly rate for Altra use (advanced notice IS required for complex set-ups).

2. All Altra and LSC operation is by Core Facility personnel.

3. Charge will apply if user does not supply disk.

4. Set-up cost for "clean-sort" will be $150 (advanced notice IS required).

5. Assistance is available for development of flow cytometric assays and procedures upon request, and prior arrangement (any reagent or incidental costs are the responsibility of the user).

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