Frequently Asked Questions (FAQs)
What is flow cytometry?
Basic information on the theory of flow cytometry, fluorochromes, panel design and analysis is available from a variety of sources. Many useful sources are provided from vendors. While the Flow Cytometry Core Facility does not endorse any specific vendors as a reagent supplier or educational resource, a few useful websites are listed here to help get you started.
How do I plan a flow cytometry experiment?
In addition to the introductory information presented in the above question, spectral viewers and panel design tools are available on line. While the Flow Cytometry Core Facility does not endorse any specific vendors as a reagent supplier or educational resource, a few useful websites are listed here to help get you started.
Spectral viewers are particularly useful tools for panel design, in which you may view the excitation and emission of potential fluorochromes in the context of the specific laser lines and detector filter sets of the instrument you will use. Some common spectral viewers are available at the following vendors’ websites:
When designing panels, we suggest you speak with flow cytometry facility staff, as well as vendors’ technical advisors. You may also find useful panel design tools, which allow you to build a panel and source antibodies from multiple vendors. One such site, which contains the configurational information of our instruments may be found at the link below.
Additionally, there are tools for finding antibody usage data, which can help you select the best antibody to use in your panel. For example, visit the website in the link below to review antibody choices.
How should my samples be prepared?
Samples to be sorted must be prepared just prior to running on the instrument. They must be a single cell suspension and filtered through a 40 micron sieve after the last wash and resuspended in sort buffer. Samples are generally best brought on ice. See the sorting application form [PDF-192KB] for more information.
Samples for acquisition should also be a single cells suspension, usually suspended in 300-350 microliters. These samples should be filtered through a 35-40 micron strainer as well, with particular attention paid to tissue digests. Most samples are fixed in 1% PFA. If samples will not be read immediately and tandem dyes are used it is recommended to fix for 1 hour, wash away fixative and resuspend in PBS.
How do I schedule an appointment?
On instruments booked by trained users, bookings may be made independently via the online instrument schedulers. Planning well in advance will render a much better chance of getting an available time slot when needed. Instruments are often heavily used and all users rely on schedulers to plan their experiments. As a result, you can be charged for a deleted booking cancelled within 24 hours of the intended use.
For flow staff operated instruments, booking will depend on both staff and instrument availability. We do our best to accommodate users’ needs, however we require at least 72 hours’ notice and will do our best to find a time that suits the user and the operator. Cancellation with less than 24 hours’ notice can result in invoicing for the booked slot.
We ask that all users of our facility do their best to book only the time they need to acquire or sort their samples, thus freeing up instrument time for other users. As always, please communicate with the flow cytometry staff as soon as possible if there is a problem regarding your booking.
Do I need to be present during the sorting/acquisition of my samples?
We prefer sorting customers to join us for the beginning of their sorts, while gating strategy, etc. is applied. In most cases, you may leave after this step as the sort may proceed for some time.
Acquisition customers may arrange to leave their samples and information with the flow cytometry staff and do not need to be present during acquisition.
Where can I bring/leave my samples?
Samples can be left with flow cytometry staff in the LSRI, room N-107, between 9am-5pm most days. Call ahead to be sure we will be present to receive your samples. In some cases, you may leave samples finished in the evening in the Tupper sub-basement SB-C1 lab refrigerator for an early acquisition the following day. Always communicate with the flow cytometry staff about when you will drop off samples and where you will drop them off.
How much will it cost?
See fees for current rates.
There is a standard setup fee plus an hourly charge. The average sort of 10 million or less starting cells will take about 1 hour. More complex sorts, or those with significantly larger numbers of starting cells will take accordingly longer roughly 3-3.5 hrs per 300 million cells.
Samples acquired on the BD FACS Calibur may take about 1-2 hr for 30-50 tubes, depending cell concentration and required events to be recorded. Acquisition on the Fortessa due to the nature of the complexity of setup and necessary controls may be about double that of the Calibur. Rates at the Fortessa vary depending on independent vs operator assisted use.
How are results reported?
All data is provided as FCS (flow cytometry standard) files compatible with post acquisition analysis software. Although limited help with data analysis may be provided in an advisory capacity, users are responsible for their own data analysis as the facility is not equipped with resources to accommodate this part of the process. Data is currently provided on DVD to limit viral and software problems due to internet connectivity or USB flash drives.