Flow Cytometry

Flow cytometry measures and analyzes the characteristics of single particles, normally cells, as they move in a stream and are passed through a laser. Thousands of cells can be analyzed by a flow cytometer in a single second. Among the measurements derived from flow cytometry are the size, relative fluorescence and complexity of the particle.

Please read below to learn more about the services offered on our Jupiter, Florida campus. 

Flow cytometry is one of the core scientific services available on our Jupiter, Florida campus.

About

Normal operating hours: 9:00 am – 5:00 pm ET, M-F

End-user access available 24/7 after training.

Flow cytometry measures phenotypic, biochemical and molecular characteristics of individual cells or particles suspended in a fluid stream. The cells flow single file past focused laser beams and light scatter or fluorescence data is collected. Tens of thousands of cells can be interrogated by a flow cytometer in a single second. Flow cytometry can be used for a variety of applications, most commonly including cell analysis or high speed cell sorting. The Flow Cytometry Core (FCC) on the Jupiter, Florida campus of UF Scripps Biomedical Research serves the campus community as well as researchers from outside UF Scripps Biomedical Research on a fee-for-service basis.

Technologies Available:

Cell Sorting

  • ARIA III: violet, blue, green and red lasers
  • ARIA FUSION: violet, blue, green and red lasers

Cell Analysis

  • LSR II: ultra-violet, violet, blue, green and red lasers
  • GALLIOS: violet, blue, green and red lasers
  • Aurora: violet, blue and red lasers
  • ImageStream MKII: violet, blue, green, red, and side-scatter lasers

Laser Microdissection and Fluorescence Imaging 

  • LEICA LMD 7000

Hematology

  • HEMAVET 950FS

Services

Cell Sorting

FCC facility staff will perform all sorting on the BD FACSAria™ III and BD FACSAria™ Fusion. All new users are required to have an initial consultation with facility staff prior to use of the cell sorter, and all users are required to fill out a sort request form prior to each experiment. If necessary, biocontainment and biosafety concerns must be addressed by the UF Scripps Biomedical Research Biosafety Office.

A discussion on sample preparation, appropriate buffer conditions, collection tubes, instrument controls, experimental controls, etc., will be part of the initial consult required for first time users.

View our Cell Sorting Guide

Sorting on the BD FACSAria™ III

The BD FACSAria™ III is a four-laser system configuration. The FACSAria™ III uses a 488 nm (blue), 561 nm (yellow-green), 633 nm (red) and a 407 nm (violet) laser system. Sorting is available using up to sixteen fluorescence parameters as well as light scatter discrimination (cell size, organelle composition and density, and doublet discrimination). Applications include high speed, four-way cell sorting, cell purification, slide sorting and cell cloning (by automated deposition into 6, 12, 24, 48, 96 well plates). Default and optional configurations for different fluorescence parameters and combinations can be viewed here.

ARIA Instrument Configuration

Sorting on the BD FACSAria™ Fusion

The BD FACSAria™ Fusion is a four-laser system configuration. The FACSAria™ Fusion uses a 488 nm (blue), 561 nm (yellow-green), 640 nm (red) and a 405 nm (violet) laser system. Sorting is available using up to sixteen fluorescence parameters as well as light scatter discrimination (cell size, organelle composition and density, and doublet discrimination). Applications include high speed, four-way cell sorting, cell purification, slide sorting and cell cloning (by automated deposition into 6, 12, 24, 48, 96 well plates). The FACSAria™ Fusion is enclosed in a ClassII Type A2 Baker biosafety cabinet consistent with BSL2 containment.

Fusion Instrument Configuration

Analytical Cytometry

Analytical applications include immuno-phenotyping, cell cycle analysis (DNA content, BrdU or EdU) and calcium flux (ratiometric imaging), among others. Additional modalities (FRET, intracellular organelle staining, etc.) can be developed in response to need; please talk to us about your specific application.

The Core provides four different analyzers for use: a five-laser BD LSR II, a four-laser Beckman Coulter Gallios, a five-laser Cytek Aurora and a four-laser Luminex ImageStream. Facility staff can assist users in deciding which analyzer is best for their given applications. Cost recovery is based on laser configuration (more lasers = higher cost; see Pricing).

Services are provided on two levels:

  1. End-user analysis: Training is available and is encouraged for routine users. Facility staff must authorize each new user, even if they have previously trained at other institutions.
  2. Operation by facility staff: For infrequent users, it may not be useful to learn the software and hardware principles. Consequently, facility staff may provide assistance (at standard charge-back rates) for these users, based on staff availability. To request an operator-assisted analysis please use the Analysis Request Form.

All independent users are expected to follow all startup/shutdown procedures as well as Core Facility rules.

BD LSR2

The BD LSR2 is a five-laser configuration (UV 355 nm, violet 407 nm, blue 488 nm, yellow-green 561 nm and red 640 nm lasers). Analysis of up to eighteen fluorescence parameters is possible, as well as visible light scatter discrimination for cell size, organelle composition or density, and doublet discrimination. The LSR2 also features a High Throughout Sampler (HTS) for a fully automated sample acquisition. The HTS is compatible with 96- and 384-well plate formats.

LSR II Instrument Configuration and Standard Fluorochromes

Beckman Coulter Gallios

The Beckman Coulter Gallios is a four-laser configuration (violet 405 nm, blue 488 nm, yellow-green 561 nm and red 638 nm lasers). Analysis of up to 10 fluorescence parameters is possible, as well as visible light scatter discrimination for cell size, organelle composition or density, and doublet discrimination. The Gallios also features a 32-tube carousel loader that allows for automated tube sampling and analysis.

Gallios Instrument Configuration and Standard Fluorochromes 

Cytek Aurora

The Cytek Aurora is a five laser, 64 detector, full spectrum analyzer capable of analyzing panels of 40 colors or more. It features the ability to load individual tubes, a tube rack, and 96 well plate reading options. It’s unique design allows for the storage of individual single color controls for continued use up to 1 month after their initial recording as long as the instrument is QCing well.

Aurora User’s Guide


Aurora Configuration

Luminex ImageStream MkII

The Luminex ImageStream MkII imaging flow cytometer utilizes a 20x, 40x or 60x objectives to capture images from its five-laser configuration (violet 405nm, blue 488nm, yellow-green 561nm, red 642nm, and side scatter 785nm lasers). Analysis of up to twelve parameters is possible, which may include or exclude side scatter discrimination. 

ImageStream Instrument Configuration and Standard Fluorochromes

Data Analysis

FlowJo and FCS Express

The Core offers access for FlowJo and FCS Express software for post-acquisition analysis, on-site or off-site. Both offers several additional analytical features that are not available within the acquisition software installed on the instruments.

Users requiring frequent use of FlowJo or FSC Express software may purchase a FlowJo portal license which you can request from the UF ICBR Core Facility in Gainesville. Check https://biotech.ufl.edu/lab-services/ to purchase individual site licenses for either software package.

If you require a full, independent FlowJo license, please contact TreeStar Software at flowjo.com

Users who do not require frequent use of FlowJo software may borrow a USB dongle license from the Core for 24hrs only at no charge. There is a $50 charge per day for additional days, if the dongle is not returned within 24hrs.  Loss of the dongle will obligate the user to purchase a replacement.  Don’t lose the dongle!!!

Data can also be analyzed for free on the core’s user workstation using either software package. Please contact us if you have any questions or concerns.

IDEAS 6.2

The Core offers free access to IDEAS 6.2 for analyzing raw image files acquired on the ImageStream MKII. Email the flow core staff to obtain your own license or for larger files, the software is available for use on the core’s user workstation. 

Microdissection and Imaging

Laser Capture Microdissection

The Leica LMD 7000 laser capture microdissection microscope has 4X, 6.3X, 10X, 20X, 40X, and 63x objectives, automated motorized scanning and positioning, and remote stylus operated input. The microscope is also capable of cutting on any single fluorescence parameter representing commonly used fluorochromes and reporters (CFP, GFP, mCherry, etc). 

Laser capture microdissection allows for the precise and contamination-free isolation of specific areas of tissue from single cells, cell groups, or tissue regions. Dissected material is deposited into microfuge tubes for DNA, RNA, or protein recovery. The dissected material is then directly accessible for further analysis.

Fluorescence Imaging

The Leica LMD 7000 microscope has 4X, 6.3X, 10X, 20X, 40X air, 63X, and 40X oil objectives and is fitted with a DFC7000 GT monochromatic camera to image fluorescent parameters emitting in the Red, Green, and Blue channels. The latest software platform, Leica Application Suite (LAS-X), is an intuitive user interface to obtain quality fluorescence images.

Luminex ImageStream MkII

The Luminex ImageStream MkII imaging flow cytometer utilizes a 20x, 40x or 60x objectives to capture images from its five-laser configuration (violet 405nm, blue 488nm, yellow-green 561nm, red 642nm, and side scatter 785nm lasers). Analysis of up to twelve parameters is possible, which may include or exclude side scatter discrimination. 

ImageStream Instrument Configuration and Standard Fluorochromes

Hematology

Hemavet 950 FS

The Hemavet 950 FS is an animal blood cell counter and differential analyzer. The Hemavet allows for a fast and complete five-part white blood cell differential, red blood cell analysis and platelet analysis. The Hemavet is fully automated and simple to operate. Whole blood analysis is available for mouse and rat blood specimens with as little as 30 ul. Tissue culture counting is also available.

* This unit is currently in long-term storage but can be reinstalled per request

Scheduling

The Flow Core uses an online calendar system (SuperSaas) that offers better scheduling solutions, allows you to view all calendars in one location, and prevents double booking. 

Flow Cytometry Calendar

You may view the calendar at anytime, however, in order to add a new reservation, you must have an account (Create your account here). This should be completed before your training appointment. If you have been trained but have not used the core in the past year, your account may be deactivated. Contact the flow core staff to schedule your appointment and redisplay proficiency.

Reserving time on the Cell Sorter

Scheduling Policy: The final schedule is issued each Monday for the second following week. While FCC staff will make every effort to accommodate all requests, please note that submission of a request does not guarantee the requested date and time, since we may have multiple requests for the same time slots. In the event such a conflict, FCC staff will contact the involved parties to try to accommodate all parties. If this is not possible, FCC staff will prioritize the requests based on special circumstances and/or the relative amount of recent usage by the requesting individual and/or his/her lab. Once sort requests are approved, an email confirmation will be sent, confirming the date and time. For time sensitive experiments, we recommend that you do not start your procedure until an appointment is confirmed.

Once the weekly schedule has been posted on Monday, all remaining unassigned time is open for booking on a first-come, first-served basis.

Note that a biosafety assessment must be conducted for each new type of sort request.

After-hours Sorting: The Flow Core recognizes a need for cell sorting experiments which fall outside of business hours due to special circumstances. To better facilitate these experiments, the Flow Core will accommodate cell sorting until 7:30pm, Monday through Thursday. Special circumstances which may warrant this accommodation include:

  • Late receipt of live samples from external sources which require immediate processing.
  • Exceedingly long sample preparation methods that must begin in the wee hours of the day.
  • All other special accommodations should be communicated to the Flow Core.

Special circumstance requests should be submitted via the sort request form with a minimum of one week notice. Sort requests received with less than one week notice will be accommodated dependent on operator availability at their sole discretion.

Delays or changes: Please note that we cannot delay the appointments of others if you are late for your sort. If no one is scheduled, late sorts or additional sort time will be accommodated within the normal operating hours of the facility. You must notify the Flow Core by email SCRPS-FlowCore1@mail.ufl.edu as soon as possible if there are any changes to the biosafety background information and/or the cell sorting details (ie. sample number, cell number, staining panel). 

Reserving time on the Analyzers

  • If you are not an authorized user, please contact the FCC at SCRPS-FlowCore1@mail.ufl.edu for scheduling and/or training.
  • Independent users are required to follow all startup and shutdown procedures at all times.
  • During peak hours, 9am-6pm, authorized users may schedule a maximum continuous block of 3 hours in advance. If the instrument is not booked by another user at the time of use, the user may continue operation.
  • During peak hours, 9am-6pm, users may schedule more than one appointment per day with a minimum of 3 hours between appointments. Exceptions may be made for special circumstances. Please contact the FCC at  SCRPS-FlowCore1@mail.ufl.edu.
  • Unfortunately, users who are late to their scheduled appointment cannot assume they will get more time. Please keep in mind your appointment may be cut short if there are other users scheduled. If users need more time, the user may continue operation depending on availability, however, the PI will be charged for the original start time through the actual end time.
  • To preserve function of the instruments, if no one is scheduled immediately after you (within 3 hours after your calendar end time), you are responsible for turning off the analyzer. Failure to comply will result in billing for the entire time that the instrument is left on and unattended. Users will also be billed at operator assisted rates for any additional time necessary to restore instrument to proper working order.
  • Modifications can be made to your reservation up until 1 minute prior to the scheduled start time.
  • Cancellations should be deleted from the calendar but are subject to a 24 hour cancellation policy. All cancellations made more than 24 hours prior to the appointment start time will not be charged. Cancellations made within 24 hours from the start time will be billed for 1/2 the original reservation time. All no shows will be billed the full reservation time. All users will get 2 grace cancellations per person, per year, with email sent to  SCRPS-FlowCore1@mail.ufl.edu.
  • If a cancellation is made, ensure the instrument was not left on for you and if so, you are responsible for turning it off. Failure to comply will result in billing for the entire time that the instrument is left on and unattended.
  • Users are responsible for exporting their data onto their own hard drive upon finishing experiments. Experiments older than 30 days will be periodically deleted from browser software without notice to the user.
  • No files should be stored on the computer’s C: or D: hard drive. Any files found on the computer hard drive will be deleted immediately without notice.

Reserving time on the Leica LMD 7000 or ImageStream MKII

Authorized users may schedule their own time.

If you are not an authorized user, please contact the FCC at SCRPS-FlowCore1@mail.ufl.edu for scheduling and/or training.

Reserving time on the Hemavet

Contact the flow core staff for use of the hemavet.

Pricing

The hourly rate is the same for all users, within or outside of Scripps, from academia or industry.  However, payments from funding sources for which indirect costs have not already been paid to Scripps will incur an additional fee for overhead cost recovery, currently 85% for academic users (inside or outside of Scripps) and 99% for non-academic use.

There is a minimum charge of 1 hour for the cell sorters, but there are no other setup charges, regardless of configuration.  Cancellations more than 48 hours before the scheduled sort time will not be charged.  Cancellations 24-48 hours in advance will be charged for one hour of time, and cancellations 0-24 hours in advance will be charged for 50% of the time reserved but minimum 1hr.  Late shows or no shows incur the full charge for time reserved.

Charges for all instruments (except the Hemavet) are determined by the time used or reserved, whichever is greater. We therefore encourage you to carefully consider how much time you reserve/request. Reservations on the analyzers are limited to a maximum of three contiguous hours.  If you need assistance in determining how much time you need, please contact us.

  • ARIA3 Cell Sorter: $60/hour (operator assisted)
  • Fusion Cell Sorter: $60/hour (operator assisted)
  • LSR2 Analytical Cytometer: $60/hour (operator assisted) $45/hour (user operated)
  • Gallios Analytical Cytometer: $60/hour (operator assisted) $40/hour (user operated)
  • Aurora Analytical Cytometer: $60/hour (operator assisted) $50/hour (user operated)
  • ImageStream MkII Imaging Cytometer: $60/hour (operator assisted) $45/hour (user operated)
  • Leica LMD 7000 (microdissection and imaging): $60/hour (operator assisted) $40/hour (user operated)
  • Hemavet 950 FS: $5/sample
  • BD 12x75mm tube with cell strainer cap (25 tubes/bag): $25.00/bag

Cytometry Information

Flow Cytometry Basics Guide: Practical Advice to Get You Started in Flow Cytometry

MiSet RFC Standards: Defining a Universal Minimum Set of Standards Required for Reproducibility and Rigor in Research Flow Cytometry Experiments

Contact/Staff

Users with Questions or Concerns should contact us using this link SCRPS-FlowCore1@mail.ufl.edu

Melissa Kazantzis

SCIENTIST
Phone: (561) 228-2898

Emily Fox

Flow Cytometry Operator I
Phone: (561) 228-2000

Scott Simanski

Flow Cytometry Operator II
Phone: (561) 228-3289