Flow Cytometry

Welcome!

It can not be overstated how valuable a face to face conversation can be when initiating a flow cytometry project. This website has some “need to know” information, but please don’t hesitate to contact James Marvin for free consultation.

The Flow Cytometry Shared Resource Laboratory offers sample preparation in addition to quantitative, multiparameter fluorescence analysis and cell sorting services. Cell sorting and analysis services are provided by core staff and/or self run options are available 24/7 following appropriate training.

Available Instruments

Cell Sorters:

  • BD FACSAria (5 laser) UV, 405nm, 488nm, 563nm and 635nm lasers, 18 colors, 4 way sort, 96 well single cell sorting
  • BD FACSAria (2 laser) 488nm and 635nm lasers, 7 colors, 4 way sort, 96 well single cell sorting (405nm and 563nm lasers coming in March!!!!)
  • Propel Labs Avalon 488nm and 563nm lasers, 4 colors, 2 way sort

Cell Analyzers:

  • BD Fortessa UV, 405nm, 488nm, 563nm and 635nm lasers, 18 colors, HTS
  • BD Celesta 405nm, 488nm, 640nm lasers, 12 colors, HTS
  • BD Canto 405nm, 488nm, 563nm and 635nm lasers, 8 colors, HTS
  • Cytek DXP 405nm, 488nm, 640nm lasers, 8 colors
  • BD Facscan 488nm, 640nm lasers, 5 colors

 

*This is a designated Huntsman Cancer Institute CCSG – Shared ResourceFor protocols, presentations, instrument specs, etc please visit http://utahflowcytometry.wordpress.com/

If you would like to make a reservation to use our equipment or services, please go to resource.cores.utah.edu.

ALL MANUSCRIPTS AND GRANTS PRESENTING WORK SUPPORTED BY THIS CORE SHOULD INCLUDE THE FOLLOWING ACKNOWLEDGEMENT

This work was supported by the University of Utah Flow Cytometry Facility in addition to the National Cancer Institute through Award Number 5P30CA042014-24.

If you used the BD FacsAria this acknowledgment is also incredibly helpful for the university.

Research reported in this publication was supported by the National Center for Research Resources of the National Institutes of Health under Award Number 1S10RR026802-01

The expertise and instrumentation to perform most flow cytometric assays that have been described in the literature is available within the expertise of the collective personnel and the physical resources of the Flow Cytometry Core Facility. One of the missions of the Core Facility is to provide access to the breadth of more complex cytometry assays that investigators would not otherwise have and to work collaboratively to bring any assay needed on line. The services provided fall into several categories, the total of which illustrates the value and extent of services offered to the community of researchers.

The assays offered by the facility range from routine cell cycle analyses and immunophenotyping to complex multi-laser applications and high speed cell sorting. Examples of the assays available include, but are not limited to the following:

  • DNA content/cell cycle measurement, including the simultaneous measurement of multiple fluorescent probes if required.
  • Immunofluorescence analyses (three to six color analyses are routinely performed, greater than seven color are under development) including characterization of plasma membrane antigens/receptors, cytoplasmic proteins, and nuclear antigens.
  • Characterization of cell populations based on scattered light intensity measurements, and autofluorescence.
  • Cell sorting including viable, sterile cell sorting and spot blot analyses.
  • Intracellular calcium flux
  • A range of apoptosis assays including Annexin V, TUNEL, mitochondrial membrane potential, and measurement of proteins involved in apoptosis regulation such as multiple members of the BCL-2 family of proteins, CD95 and caspase protein levels/activity
  • Fluorescence Resonance Energy Tranfer (FRET)
  • Nanoparticle characterization
  • Bivariate and univariate chromosome analysis
  • Receptor-ligand interactions
  • Cell proliferation studies including BrdU incorporation and CFSE tracking
  • Viability assays (membrane exclusion and metabolic viability).
  • Various function assays including oxidative metabolism, neutrophil function (oxidative burst, phagocytosis), cytoplasmic pH, membrane potential.
  • Kinetic analyses
  • Signal transduction pathway analyses (simultaneous assessment of multiple intracellular phosphorylated epitopes combined in complex multi-color assays)
  • Sample preparation and staining

Full service: consultation and training

The Flow Cytometry facility is unique in the fact that it offers investigators the entire spectrum of  cytometric experiment management, if desired, all the way from initial design consultation to the creation of graphics for publication.  It is entirely up to the investigator what  level of involvement the facility staff takes.  The staff will prepare samples including staining, data collection, quality control, data analysis/interpretation, and creation of graphics.  Alternatively, if the investigator chooses the facility will solely provide consultation on any of the above so that the research is entirely in the hands of the investigator.  Sample preparation provided by the flow lab personnel has proven to be extremely valuable in that a subset of investigators may lack technologist time, or expertise, to carry out properly designed flow experiments. This service has been particularly helpful to more clinical orientated investigators allowing expansion of their laboratory based studies.

Cell Sorting
Data Analysis

Software available in the Analyzer Lab (Wintrobe Rm 603) includes:

  • FlowJo (visit http://utahflowcytometry.wordpress.com/faqs/ for information to get involved with University Site license)
  • Becton-Dickinson’s CellQuest, the basic data acquisition and analysis software used on the FACScans and Vantage SE sorter
  • ModFit, a DNA/Cell Cycle modeling program from Verity Software House
Instrumentation

**Note: External academic prices are 1.5x the listed price while external industry prices are 2x the listed price.

BD FACSAria Cell Sorter

Optical Configuration and Publication Information
Location: Wintrobe Bldg Rm 638
Features:

  • 5 Lasers (UV,405,488,561,640)
  • 13 color detection
  • 96 well collection

U of U Price: $87.59 (non-sterile); $112.81 (sterile)

Make an Appointment

 

Propel Labs Avalon Cell Sorter

Optical Configuration and Publication Information
Location: Wintrobe Bldg Rm 638
Features:

  • 2 Lasers (488,561)
  • 2-way sort
  • 4 color detection

U of U Price: $68.42 (non-sterile); $60/hr (sterile)

Make an Appointment

 

lsr-fortessa-hts

HCI Fortessa Analyzer

HCI Fortessa Operation Guidelines

HCI Fortessa Configuration

Location: HCI RM 4251 Contact flow lab
Features:

  • 4 Lasers (405,488,561,640)
  • 16 color detection
  • 96 well High throughput sampler

U of U Price: $29.00 per hour

Make an Appointment

 

BD FACSCanto Analyzer

Optical configuration and Publication information
Setup and usage instructions
Location: Wintrobe Bldg Rm 603
Features:

  • 4 Lasers (405,488,561,640)
  • 8 color detection
  • 96 well High throughput sampler

U of U Price: $29.31 (user run); $78.13 (Core run)

Make an Appointment

 

Cytek DxP Analyzer

Optical configuration and Publication information
Location: Wintrobe Bldg Rm 603
Features:

  • 3 Lasers (488, 405,640)
  • 8 color detection

U of U Price: $25 (user run); $78.13 (Core run)

Make an Appointment

BD Celesta

Location: Wintrobe Bldg Rm 603
Features:

  • 3 Lasers (488, 405,640)
  • 12 color detection

U of U Price: $25 (user run); $78.13 (Core run)

Make an Appointment

 

BD FACScan Analyzer

Optical configuration and Publication information
Setup and usage instructions
Red Laser instructions
Location: Wintrobe Bldg Rm 603
Features:

  • 2 Lasers (488, 640)
  • 5 color detection

U of U Price: $20 (user run); $40 (Core run)

Make an Appointment

 

Offline Analysis Workstation

Location: Wintrobe Bldg Rm 603
Features:

U of U Price: Free

Make an Appointment

How can I start using your facility?

In order to get access to the scheduler you will need to fill out the Work Authorization Form. Once complete, you can return to the address listed at the top of the form. I normally like to do instrument training with actual samples. That way we can tailor the training specific to your assay and set up a template and instrument settings with you. Once you know when your cells will be ready send me an email jmarvin@cores.utah.edu and we will set up the appointment.

How can I get access to facility instruments after hours?

Once you are fully trained on a particular instrument you are eligible for after hours access. In order to get your ID card activated you need to go to the core facilities administration office located at SOM 5C124. Jeff Ware (3-2926) or Esther Kim (3-2928) will need your ID number and also the 6 numbers following the 2* on the back of the card

How can I get involved with the FlowJo Site License?

Please follow the steps below to register your computer:

  1. Install Flowjo on a computer. Under the “Download” tab, you can get to “legacy” versions or Version X for Windows or Mac. If you are using windows I recommend the newest Version X. If you are using a Mac I still really like the Version 9 or you can use the new Version X. Both downloads are available on that page. And you can have both versions downloaded if you want. Please contact me if you are unsure which version you want to use. Once it is installed it will ask you to install a serial number after you agree to the license agreement. At the bottom of that screen will be the hardware address that needs to be registered.
  2. To register, follow this link. Do not add spaces or colons when you enter your Hardware address.
    Once registered, install the serial number R596cZ4aYgpE9554. Once the serial number is installed, the program should work immediately. There may be issues with your firewall or proxy server (if you have one) at first. If you experience any problems using the software after registration please call or email me and I will get it worked out for you.

PRICING!!! We will hopefully have enough copies at the end of the year so that it comes out to about $250/yr. However they bill quarterly, so if you only use it for 1 month of the year, you only get billed for 1/4 of the full price.
Basically how this works is that they send me a bill once a year and then I pay them the full amount. Then go out and collect from the registered users.

Please feel free to contact me with any questions that you may have.

Which cell sorter should I use?

Well thanks for asking. As you noticed we have two cell sorters, a BD FacsAria, and a Propel labs Avalon. The BD FacsAria is going to be more appropriate for multicolor hematopoietic type sorts. For example subsets of Tcells from the spleen or bone marrow. Although we have multiple sized nozzles for the Aria, we routinely run the 85um nozzle with about 40psi of pressure. This pressure has been shown to cause viability and growth issues with some larger more fragile cells. The Avalon is more limited in capabilities. It only has two lasers and 4 color detection. So this instrument is more ideally set up for larger cells transfected with GFP, RFP or other similar proteins. The Avalon has a 100um nozzle and runs with a lower pressure of 30psi. Therefore it is more gentle on the cells. Another key difference between the two instruments is how many populations can be collected at once. The Aria can sort 4 populations at once, and the Avalon can only collect 2 populations at once. Also, the avalon can only collect into eppendorf or 5ml tubes. The Aria can sort into eppendorf, 5ml, or 15ml tubes or 96well plates.

What do I need to bring to sort cells?

slide1

 

  1. Samples – Your samples can be in 5ml tubes or 15ml conical. You should aim for a concentration around 10 million/ml for cell lines or 15 million/ml for lymphocytes. If you don’t have nearly that many cells, don’t resuspend in less than 500ul. Buffer can be anything that the cells are happy in. More detailed info here
  2. Controls – For fluorescent protein work we need an untransfected sample to set up the instrument. For multicolor experiments we need unstained in addition to single color controls.
  3. Collection Tubes – We can collect into eppendorf, 5ml, 15ml 50ml or 96well plates. Each collection tube should have collection media already in the tube.
  4. Extra media – In case there is a clog and a sample needs to be resorted.
  5. Jump Drive – If you need your data files
  6. Ice – If appropriate.

How should I acknowledge the facility in my publication?

The most important acknowledgement for the facility and university is referencing the shared instrumentation grant # IF YOU USED THE CELL SORTER for any work related to your publication. Something like this would work.

“Research reported in this publication was supported by the National Center
for Research Resources of the National Institutes of Health under Award
Number 1S10RR026802-01″

Also referencing the NCI Award Number 5P30CA042014-24 is helpful.

 

Hours of Operation

Please call the lab at 801-581-8641 or the director 801-585-7382 for hours of operation.

Location

Maxwell Wintrobe Research Bldg (Bldg 530), Wintrobe Rm 603 & 638
26 N Medical Dr.
Salt Lake City, UT 84112
Office: 801-585-7382 | Lab: 801-581-8641

Staff

James Marvin, Director
jmarvin@cores.utah.edu
Office: 801-585-7382
Lab: 801-581-8641
Wintrobe Bldg, Rooms 603 & 638
http://utahflowcytometry.wordpress.com/

Oversight Committee

Ryan O’Connellryan.oconnell@path.utah.edu 801-213-4153
Thomas O’HareThomas.OHare@hci.utah.edu 801-585-0256
Gerald Spangrudejerry.spangrude@hsc.utah.edu 801-585-5544
Matthew Williamsmatthew.williams@path.utah.edu – 801-585-2487