The CyTOF Mass Cytometer is the latest in technology and high end instrumentation from Fluidigm (formally DVS Sciences). This instrument functions similar to traditional flow cytometry except for using metal in place of fluorescence to tag antibodies. With the use of metal-ion tagged antibodies, extremely high multiparametric analysis (30-40+ simultaneous parameters) can be done on your samples.
For a general overview of mass cytometry, please read this presentation. There is additional info in the Library share of the server for anyone interested in more information. Go to the Registration & Training page for instructions on how to connect.
General Information before running samples
- Before samples can be submitted to the Flow Core for analysis, an initial meeting with including the PI and members of the PI’s lab who will be doing the labeling, the FCC (technical considerations) and the Rochester Human Immunology (biological considerations) is required. This will ensure we have the best success with adopting this new technology. We have set aside Tuesday’s at 12 noon in 3-4103 as a standard time for this meeting. Other times can be made by appointment. Please contact Matt Cochran if you'd like to schedule a time.
- Review the reagents listed/available at the Fluidigm website.
- Labeling Protocols - Protocols for labeling are in the Flow Core Server Library.
- Data Analysis in mass cytometry experiments can be performed using any flow cytometry analysis software using traditional gating methods. In addition, most analysis packages including Flowjo now include additional tools to deal with the highly multiparametric data from mass cytometry panels. For a discussion of advanced analytical tools like clustering and dimensionality reduction algorithms please contact the Matt Cochran.
- Sample Preparation - There is specific information a user needs to be aware of when planning how samples need to be submitted for analysis
- Sample Concentration - samples need to be no more than one million cells per ml. If the concentration is higher, data quality will suffer.
- The sample can be run via the Supersampler (preferred for larger continuous volumes) or manually via separate pushes of 500uL (rarely used).
- Sample Running Duration - The instrument has a fixed sample introduction rate of 45uL/min meaning that 1mL of sample will run for ~23 minutes via the Supersampler.
- Individual 500uL sample pushes will take ~11 minutes, not including washing (3-10 minutes).
- Barcoding of samples - Samples can be batched and barcoded via reagents available through Fluidigm which will reduce staining variation, improve throughput on the instrument, and reduce any contribution of instrument variations. For these reasons we recommend barcoding of samples when possible.
CyTOF Submission Form
Please fill out the CyTOF Request Form. The username and password are fcuser, all lowercase.
Please allow a minimum of 72 hours advanced notice. The staff will do their best to accommodate all requests but do not begin the experiment until a time is officially scheduled.
After submission of the CyTOF Request Form, the following information should be taken into account:
- Concentration of samples should not be more than 1 million/mL. Data quality can suffer at higher concentrations.
- Know your sample volumes and how you would like your sample to be introduced to the instrument.
- How many events do you need for analysis? Average recovery is ~40% of sample input.
- Know your panel: Be prepared to double check the template.
- All samples must be washed and resuspended in ultrapure water.
- All samples will be filtered by FCC staff prior to the run.
- Late arrival of samples could result in having to postpone the run and impact data quality.
- Bring samples in locking secondary containment with biohazard label.
If you have trouble filling out the form, please send us an email or call 276-5330 and ask for Terry, Sharleen, Julia or Matt.
The CyTOF Team
Matt C 3-4020