Flow Cytometry

In addition to cell sorting, acquiring, analyzing, archiving, and preparing flow data for publication, the FSCR staff provides training of the analyzers and image cytometer for self-users, and consultation, technical advice, collaboration, and technical information for potential users. The FCSR staff has experience in nearly all areas of flow cytometric analysis and cell sorting applications and strives to help investigators develop new applications. Staff members recommend assays, help to develop and troubleshoot new protocols, participate actively in data analysis, and, in general, work closely with investigators to fine tune their individual experiments. An important part of the mission of the FCSR is the education of customers to insure all users and their lab members get the most out of the technical resources available. We also participate in the ongoing education of technicians, graduate students, post-docs, and MDs.

Another important function is to help investigators evaluate existing cell separation technologies before choosing flow cytometry. Thus, the FCSR staff works integrally with investigators at all levels of experience and expertise to solve problems and meet investigators' scientific needs.

The DCI Flow Cytometry Shared Resource now uses a scheduling calendar available at the Core Research at Duke website for all reservations, whether staff assisted cell sorting or self-use of our call analyzers.

It is the responsibility of the PIs and the Financial Managers to link student, post-docs, and technicians to the fund codes owned by the PIs.  An individual may be linked to one or more fund codes and can select the proper code to apply to any job performed in our shared resource.  The following link gives one access to the daily scheduling calendar for all of our cytometers: https://medschool.duke.edu/research/core-research-facilities.

Click on the leftmost blue circle and sign in with your net ID and password.

If you have questions about how to use the Core Research, the center blue circle has many how-to links for PIs, Financial managers, and facility users alike.

Please do not use the Firefox browser. It is not compatible with CoreResearch. The Core Research website is available from anywhere on or off campus with no need to VPN.

Potential self-users of the Fortessa and the Imagestream are required to first receive training.

Self-users can schedule time using the online CoreResearch calendar for all 4 of the analyzers (CANTO A or CANTO B in 343 Jones, FACSCalibur in 307 Jones, Fortessa X-20 in 369 Jones, and the Amnis Imagestream in 369 Jones.

All analyzers utilize Falcon brand 5 ml polystyrene (clear plastic) samples tubes.  Canto B can also accept any standard 96 well plate, but this requires one training session.  The Imagestream utilizes only standard Eppendorf 1.5 ml snap cap tubes.

Alternatively, customers can make appointments for staff-assisted live cell analysis by first contacting DCIflow@duke.edu or 919-613-7820.

Customers can also have staff-performed fixed cell assays performed by again first contacting Javid Mohammed. Experiments with an accompanying explanatory written key can be dropped off in room 307 Jones Bldg after signing the "Research Signin" clipboard on the counter. The fridge is under the counter. Evening or weekend dropoffs can be put in CR308, the coldroom just down the hallway from room 306.

Customers can directly make their own reservations to schedule their sorting times on the Astrios sorter (Lynn Martinek in room 336 Jones, 919.613.7821), or the DiVa sorter (Bin Li in room 307 Jones, 919.613.7818) using the online calendar. Typically after the first couple of appointments are made by telephone, a new customer is able to accurately judge how much time to reserve.

Sorting reservations on the MoFlo XDP sorter (Nancy Martin in room 336 Jones, 919.613.7836) are always made by mutual agreement with Nancy over the telephone.

When making online sorting reservations, you can utilize the "Experiment Information" field in the bottom left corner of the "Reservation Tab" by using text to briefly indicate your parameters (e.g., fluorochromes, number of samples, number of samples, 96 well plates, etc.). Cancellations prior to 48 hours before the start of the reservation can easily be made by the client using the online CoreResearch calendar. 

Cancelling Cell Sorting Reservations

Once closer than 48 hours, you can’t cancel without contacting the staff, Lynn Martinek (919.613.7821), Nancy Martin (919.613.7836), and Bin Li (919.613.7818).  If you cancel closer than 48 hours to your reserved time, you will be charged for 50% of the time. In fact you will be charged for 50% of your time even if you actually use less than 50% of the reserved time.

As soon as you realize that you can't use your scheduled time, please call and speak to one of the staff directly or email all of the staff simultaneously (to insure a rapid response). Staff performed sorting time is at a premium.

If you find a replacement or we find a replacement you will not be charged for the replaced time.

If you cancel inside 24 hours you may be charged 100% of the reserved time if no replacement sort can be found.

If you simply fail to show or call concerning a sort appointment, we will always charge you for 100% of the reserved time.

Cancelling Self-Use Reservations

There is no penalty for cancelling self-use reservations, but we strongly urge clients to book time as efficiently as possible. Please respect your fellow researcher’s valuable time as your own.

Remember-If you don’t do anything to your reservation, CoreResearch will assume that the time was used as reserved and the fund code will automatically be charged.

All of our cell sorters are multi-laser, have temperature controlled sample and collection options, biohazard control, and have a single cell deposition unit for sorting into multi-well plates.

• B-C Astrios: Purchased in 2012, this is our most versatile cell sorter. It has four lasers (405 nm, 488 nm, 561 nm, and 640 nm). It can collect up to 14 fluorescence signals and is housed inside a Baker Bioprotect III tissue culture hood for increased biosafety. It can sort up to six populations simultaneously with index sorting to identify individual cells in single cell sorting mode. It is equipped with automatic sort setup and sort monitoring functions.
• B-C Astrios. This second Astrios, acquired as a replacement for the former Moflo XDP is essentially identical to the first Astrios listed above with one major difference.  It is not housed inside a Bioproect Hood but does have a basic biohazard containment system. The lasers and detectors, index sorting, auto sort setup and sort monitoring, single cell deposition, and temperature control are all identical to the other Astrios.
• BD DiVa: This sorter was purchased in 2002 as a joint venture between the DCI, the School of Medicine, and the Department of Immunology.  It has three lasers for excitation, 488 nm, either 360 nm UV or 413 in the violet, and a HeNe laser at 632 nm. It has 14 optical detectors, the standard two for light scatter, and 12 for various fluorochromes, making it extremely versatile for multi parameter phenotyping. It is currently our only cytometer with a UV laser.

Analyzers:

• BD Fortessa X-20 (NEW NOV 2016).  This is a five laser analyzer (UV, 405, 466, 561, and 640 nm excitation) with 18 potential channels for fluorescence emission.  It utilizes the BD FACSDiVa software with which users of the Canto II instruments are already familiar.
• BD FACSCanto IIs: We have two of these three laser, eight color analyzers that were purchased in 2008 and 2010. They are available for self-use 24/7. Each has a violet laser that excites fluorochromes at 405 nm, in addition to conventional 488 nm and 633 nm lasers. They both run BD's FACSDiva software. Canto B has a 96 well plate sampler.
• BD FACSCalibur: Although this 2 laser, 4 color analyzer is used predominantly for fixed assays such as immunophenotyping and cell cycle analysis, a wide range of flow assays on both live cells and fixed cells can be performed.
• Imagestream X (Amnis Corp): The Imagestream has 4 lasers, and can record five fluorescent colors and a bright-field image. It can analyze subcellular localizations and morphologic changes on several hundred cells per second. It runs the powerful proprietary Ideas software (Amnis Corp). This is a self-use instrument after appropriate training by the staff.

Self-users are expected to back up their own data utilizing the Shared Resource space on the Medical Center's Isilon server as an intermediary short term repository prior to additional backup on your lab’s workstation. New PIs and new users must request access to the server. A work station with FloJo analysis software (V9 or V10) is available in 369 Jones free of charge.

We have only two basic rates for staff-performed or user-performed work. We then apply a 25% discount to each rate for DCI members. The standard rate for staff-performed work for non-DCI members is $2.00 per minute (~$120 per hour); that for user-performed work is $0.83 per minute (~$50 per hour). With their added discount, DCI members pay a competitive $1.50 per minute (~$90 per hour) for staff-performed work and $0.63 per minute (~$38 per hour) for user-performed work. Time is recorded and charged to the minute. We do not differentiate between cell sorting and analysis, or between data acquisition and post-acquisition analysis. We do not charge for consultation, customer education, or discussion of data with the customer. The work stations are provided to customers free of charge.