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Mass Cytometry - CyTOF - Behemoth
The CyTOF Mass Cytometer is the latest in technology and high end instrumentation from DVS Sciences. This instrument functions similar to traditional flow cytometry except for using metal in place of fluroescence 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. Please follow this link 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 DVS website.
- Labeling Protocols - Protocols for labeling are in the Flow Core Server Library.
- Data Analysis with Cytobank - We have chosen to partner with Cytobank for our data storage and analysis needs. This will be free to all users and details about signing up for the Rochester instance of Cytobank is posted in the FCC library. Go here to register for Cytobank.
- 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.
- Push Volume - each sample is pushed in 500ul increments. Samples greater than 500ul will be run in multiple pushes.
- Number of Pushes - Any sample that requires multiple pushes will be run one after another. This means a 2ml sample will be run in four consecutive pushes.
- Sample Running Duration - There are two speeds the instrument can be run at, and each has different durations as a result. Slower run speeds result in fewer lost events.
- Run Speed 0.060ml/min - Each sample takes ~15 minutes to run, cleaning and turnover time included .
- Run Speed 0.045ml/min - Each sample takes ~18 minutes to run, cleaning and turnover time included.
CyTOF Submission Form
Please follow this link to fill out a CyTOF Request Form. The username and password are fcuser, all lower case.
Please allow a minimum of 48 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. Each push is 500uL of sample so larger volumes will be run in multiple pushes, which extends length of the run.
- How many events do you need for analysis? Average recovery is ~20% 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 Cristy, Terry, Sharleen, or Matt.
The CyTOF Team
Matt C 3-4020
Location/Time - Rm 3-4103 @10am (in the flow core hallway)
Schedule: Nov 14 and 28, Dec 12
next lecture coming soon!
Spectral Viewers and Panel Design
All guidelines are available in the "User Docs" folder located on every instrument computer as well as in the FCC_Library server folder.
Comments about the Flow Core?