Self Analysis on the CyTOF

CyTOF2 Charmander

Availability: 24 hours, 7 days a week.  (Monday-Friday, 9am-6pm with "CyTOF-Setup.")

Overview:  The Fluidigm CyTOF is a mass spectrometry cytometer analyzer capable of generating data of 48 parameters for each event passing through the detector.  As more reagents are tested and developed, the goal will be over 50 parameters. 

The workflow and operation are very similar to flow cytometry, but rather than using fluorescence, elemental tags are utilized.  Analysis occurs in the Time-of-Flight (TOF) chamber, allowing detection of over 40 different parameters simultaneously. It is capable of analysis rates of up to 500 events per second.  This analysis instrument provides a more shotgun like approach and can be very useful if you have limited sample, and wanted to extract as much information as possible, allowing you to discover markers you would never thought that would be linked.  With appropriate markers, information on dynamic changes to cell populations in disease progression, therapeutic treatment, stable remission, or relapse can be compared.  With Palladium isotope barcoding, up to 20 different samples can be antibody stained within a single tube for direct comparison of expression levels.  In addition, relative mRNA transcription levels of a selected number of genes can also be analyzed.  Once markers of interest have been identified, it would be recommended to shift to more standard flow cytometry techniques to examine your cells of interest further as some markers might not be necessary.  It is operated using CyTOF Acquisition Software and is available for all labs to use.  A Super Sampler unit is available to boost sample recovery and speed up acquisition time.

Cytof workflow

The figure to the right provides a brief summary on how mass cytometry works.  A liquid sample containing cells labeled with antibodies tagged with heavy metal isotope conjugated probes (A) are introduced into a nebulizer creating an aerosol (B) which is directed towards a plasma torch (C) where the cells are vaporized.  Low mass ions are removed (D), and the ion cloud enters the TOF chamber where probes are separated based on their mass to charge ratio as they accelerate towards the detector (E).  The time-resolved detector measures a mass spectrum that represents the identity and quantity of each isotopic probe on a per-cell basis (F).  Data is generated in FCS format (G) and can be analyzed using third party software (H).

Seminars:  If you want to learn more and get some feedback about potential projects, you should join the CyTOF Interest Group Meeting, usually held on the second Wednesday of the month at 5pm in HSW1057 at the Parnassus Campus.  The event is sponsored by Fluidigm with food, drink, and technical support. Contact Annie Chan at Annie.Chan@ucsf.edu to be added to this specific listserv.

Training:  If you are a new user to this service, please setup a meeting with the Core manager, to discuss what you plan on running and panel design. All CyTOF users need to fulfill Basic Training requirements.  Click HERE.

Assistance:  If you run samples infrequently, it is recommended to request Assistance to have a Flow Core staff member run samples for you. Click HERE.

Rates:  Click HERE.

Scheduling:  Click HERE.

Setup & Operation:  During normal CyTOF working hours of Monday-Friday, 9am-6pm, the CyTOF will be setup for you by Flow Core staff. “CyTOF-Setup” is defined as: nebulizer cleaned and inserted, plasma torch fired up, machine tuned/QCd, and Super Sampler installed.  After setup has been performed, you can run samples yourself once you have completed Basic Training.  If you are not a superuser and need assistance setting up the instrument during non-normal work hours (Monday-Friday from 6pm-9am, weekends, or holidays), you can request 2 hours of Assistance from a core staff member so we can get the instrument setup for you. If you run samples frequently during non-normal work hours, it is highly recommended to go through the CyTOF Advanced Training to become a superuser.

Data Analysis:  Raw data is in a FCS file format. For analysis options, click HERE.  The Core recommends using Cytobank, which can be utilized for storing and analyzing CyTOF data as well as FCS files off any other flow cytometer.

Instrument Features:  The Summary Table HERE will provide a matrix of all the key features of each instrument.  Click on the nickname link of a specific instrument to get a more detailed layout.  Tabs are also at the bottom listing the machine nicknames.  It is essential for planning your experiments and seeing if an instrument can detect your specific markers.  If you need to download a copy, click on "File" then "Download as" from the Google menu.  Check out what the instrument is equipped with and contact us if you have any questions about it.